ES2197458T3 - TISSUE FACTOR METHODS AND COMPOSITIONS FOR COAGULATION AND TUMOR TREATMENT. - Google Patents

Abstract

The invention embodies the surprising discovery that Tissue Factor (TF) compositions and variants thereof specifically localize to the blood vessels within a vascularized tumor following systemic administration. The invention therefore provides methods and compositions comprising coagulant-deficient Tissue Factor for use in effecting specific coagulation and for use in tumor treatment. The TF compositions and methods of present invention may be used alone, as TF conjugates with improved half-life, or in combination with other agents, such as conventional chemotherapeutic drugs, targeted immunotoxins, targeted coaguligands, and/or in combination with Factor VIIa (FVIIa) or FVIIa activators.

Description

Tissue factor method and compositions for Coagulation and treatment of tumors.

Background of the invention 1. Technical sector to which the invention belongs

The present invention relates generally to fields of blood vessels and coagulation. Plus particularly encompasses the surprising finding that Tissue Factor compositions can locate the vasculature of the tumor and cause specific coagulation. They make themselves known particularly methods and compositions for effecting coagulation specific and to treat tumors with Factor compositions Tissue (FT) and combinations of Tissue Factor and others molecules

2. Description of the related technique

The resistance of tumor cells to Several chemotherapeutic substances represents a problem important in clinical oncology. Therefore, although they have made many advances in the chemotherapy of the disease Neoplastic during the past 30 years, most of the most prevalent human cancer still resist intervention effective chemotherapeutic.

A significant underlying problem that is due attacking in any treatment regimen is the concept of `` total elimination of cells ''. This concept holds that with in order to have an effective treatment regimen, either the surgical or chemotherapeutic approach or both, there must be a total elimination of cells, from the so-called malignant cells `` clonogenic '', that is, cells that have the ability to grow uncontrollably and replace any tumor mass that It could have been removed. Due to the final need of develop therapeutic substances and regimens that achieve Total cell removal, certain types of tumors have been more receptive than others to therapy. For example, tumors of soft tissue (for example, lymphomas), and blood tumors and of the blood-forming organs (for example, leukemia) have generally responded better to chemotherapeutic therapy than solid tumors such as carcinomas.

A reason for the susceptibility of tumors soft and blood-based chemotherapy is the biggest physical accessibility of lymphoma and leukemic cells to the chemotherapeutic intervention. It is simply much harder for most chemotherapeutic substances reach all the cells of a solid tumor mass that to soft tumors and blood-based tumors, and therefore it is much more difficult achieve total cell removal. Increase the dose of chemotherapeutic agents frequently results in effects toxic side effects, which generally limit effectiveness of conventional antitumor substances.

It has long been clear that there is a significant need for the development of novel strategies to the treatment of solid tumors. One of these strategies is the use of `` immunotoxins '', in which an antibody of antitumor cell to administer a toxin to the cells of the tumor. However, as with the chemotherapeutic approach described above, it also suffers from certain disadvantages. For example, antigen-negative cells or antigen deficient can survive and repopulate the tumor or lead to other metastases. Also, in the treatment of tumors solids, tumor mass is generally impermeable to molecules about the size of antibodies and immunotoxins. Therefore the Development of immunotoxins alone does not lead to improvements particularly significant in the treatment of cancer.

Certain researchers then developed the approach to target vasculature of solid tumors. By targeting the blood vessels of the tumors, it they have certain advantages because it is not likely to lead to development of resistant tumor cells or populations thereof. In addition, the administration of target substances to the vasculature does not have problems related to accessibility, and destruction of the blood vessels will lead to an amplification of the effect antitumor since many tumor cells depend on a single vessel for your supply of oxygen and nutrients. Strategies with Vascular objective by way of example are described in Burrows and collaborators (1992), in Burrows and Thorpe (1993) and in WO 93/17715. This targeted administration of anti-cellular substances to the tumor vasculature discloses strategies quite promising, however, the use of toxin parts of these molecules still leaves room for objective improvement vascular.

Another approach to the directed destruction of the Tumor vasculature has been disclosed in WO Patent 96/01653, in which antibodies against the markers of the vasculature of the tumor to administer coagulants to the vasculature of solid tumors. Targeted coagulant administration of this way has the advantage that they are not likely to result Significant toxic side effects of any fund that fails the objective that could result due to any reactivity low level cross-targeting of antibodies directed with cells of normal tissues. The constructions of coagulant antibodies for use in this therapy Targeted antitumor have been called `` coaguligands '' (WO 96/01653).

Although the specific administration of a coagulant to a vessel of a tumor marks a surprising breakthrough, the use or manipulation of coagulation in relation to treatment of various diseases and human disorders has been practiced during some time. As an example only, Morrissey and Comp have proposed the use of Truncated Tissue Factor (tTF) in combination with Factor VIIa (FVIIa) in the treatment of patients, such as hemophiliacs, in which coagulation of the blood (U.S. Patent Nos. 5,374,617; 5,504,064; and 5,504,067). Roy and Vehar have also developed tissue factor mutants which neutralize the endogenous tissue factor and can be used as anticoagulants, for example, in the treatment of heart attack myocardium (U.S. Patent Nos. 5,346,991 and 5,589,363).

In other studies concerning the Tissue Factor (FT), Edgington and colleagues have shown that, in contrast to normal melanocytes, human melanoma cells with metastases malignant express high levels of Tissue Factor, the initiator cellular major of plasma coagulation protease cascades (WO 94/28017; WO 94/05328; U.S. Patent No. 5,437,864). I know reported that the inhibition of Tissue Factor function and the subsequent reduction in the generation of local protease gave as result significantly reduced numbers of tumor cells retained in the vasculature. This leads to the suggestion that there are a direct correlation between the expression of the Tissue Factor and the metastatic phenotype of tumor cells. Edgington and collaborators proposed that a function of the Factor of Tissue for the successful implantation of tumor cells and that the interference with the function of the Tissue Factor, or the specific interference with cell surface expression of the Tissue Factor, it is useful to inhibit metastasis. These authors have therefore proposed to treat cancer with antibodies directed against the Tissue Factor.

Summary of the invention

In direct contrast to the observations Edgington and colleagues' previous and antibody uses Tissue factor to treat cancer, the present inventors have shown that Truncated Tissue Factor compositions and Tissue Factor variants can, in themselves, be used in the solid tumor treatment. The present invention was developed, in part, from the surprising discovery of the inventors that the Truncated Tissue Factor specifically locates the blood vessels within a vascularized tumor simply after systemic administration. This location in absence of any objective fraction could not have been foreseen to from previous detailed studies of the molecule of Tissue Factor The autolocalizing nature of the Factor Fabric, as described in the present description, also contrasts with the previously described uses of the Fabric Factor in the treatment of blood disorders, for example, in hemophiliacs, in which the administration and action of the Factor Tissue either not located or limited to topical application in A specific area

Therefore, in certain embodiments, the The present invention discloses methods to favor the coagulation in prothrombotic blood vessels of an animal or patient, these methods generally include administering to animal a composition comprising a compound of Factor of Poor coagulation tissue in an amount effective to promote coagulation preferably, or specifically, in the prothrombotic blood vessels

As used throughout the description, the terms `` a '' and `` one '' are used in the sense that they mean `` at least one (one) '' `` at least a first or a first '', `` one, one or more '' or `` a plurality '' of the components or stages mentioned, except in cases where a limit is established superior specifically after that. Therefore `` a Factor of Poor coagulation tissue '' means `` at least a first Poor coagulation tissue factor ''. Limits and operable parameters of combinations, as well as quantities of any single substance, they will be known by technicians with ordinary experience in the field in light of the present description.

Prothrombotic blood vessels may be associated with any of a series of angiogenic diseases, with a benign growth or with a vascularized tumor. In the context of the present invention, the term `` a tumor vascularized '' means a vascularized malignant tumor. The The present invention is particularly advantageous for treating tumors. vascularized at least about medium size and for treat large vascularized tumors.

The composition is generally pharmaceutically acceptable and preferably will be administered to the animal systemically, such as through intravenous injection.

The methods are further described as methods for treat an animal or human patient who has an associated disease with prothrombotic blood vessels, which include administering to the animal an amount of at least a first composition coagulant comprising at least a first Factor compound Deficient coagulation tissue effective for preferred or specifically promote coagulation in blood vessels prothrombotic associated with the site of benign disease or malignant

The essence of the invention can also be define as a composition that comprises at least a quantity biologically effective of at least a first Factor compound of poor coagulation tissue for use in the preparation of a medicine for use in promoting preferential coagulation or specifically in prothrombotic blood vessels of an animal, particularly those associated with a benign disease site or evil one.

In the medications and uses of this invention, one of the advantages lies in the fact that the simple disposition of the coagulant composition in the systemic circulation of the animal results in the specific location or preferential of the Fabric Factor compound with respect to the site of the illness.

Preferred methods described herein invention are for use in promoting coagulation in the vasculature of the tumor of an animal or human subject that has a vascularized tumor, these methods generally comprise administer to the animal one or more compositions comprising one or more compounds of deficient coagulation tissue factor in one sufficient quantity to specifically or preferentially promote the coagulation in the vasculature of the tumor. Tumor treatment vascularized medium or large size is particularly advantageous.

Treatment methods can be described. as methods to treat an animal that has a tumor vascularized, which comprise administering to the animal an amount biologically effective of at least one coagulant composition comprising a quantity of at least a first compound of Coagulation tissue factor deficient enough to specifically or preferentially promote coagulation in the Vasculature of the tumor

Another description is of a method to treat a animal or patient who has a vascularized tumor, which systemically comprises administering to the animal one or more compositions comprising one or a plurality of compounds of Coagulation Tissue Factor deficient in an amount or amounts and for an effective period of time to promote the coagulation specifically or preferentially in the vasculature of the vascularized tumor

The antitumor effects of the present invention are particularly described in the characterized methods, since comprise administering to the animal, with a tumor, a composition that It comprises at least one fabric factor compound of poor coagulation in an effective amount to promote coagulation in the vasculature of the tumor and for specific or Preferably cause tissue necrosis in the tumor.

These aspects of the invention also give know a composition that comprises at least a quantity biologically effective of at least a first Factor compound of poor coagulation tissue for use in the preparation of a medicine for use in promoting coagulation preferentially, or specifically, in the blood vessels prothrombotic associated with a malignant vascularized tumor of a animal; so that the medicine is therefore intended for your use in the treatment of an animal affected by cancer, causing the coagulation of the blood vessels of the tumor and necrosis of the tumor.

The terms `` preferentially '' and `` specifically '', as used in this description in the context of promoting blood clotting prothrombotic or vasculature of a tumor, and / or as used in the context of promoting sufficient coagulation to cause necrosis of the tissue at a disease site such as a tumor, means than the compound of the Tissue Factor or the combination of Factor of Tissue-second substance works to achieve the coagulation and / or tissue necrosis that is substantially confined to the site of the disease, such as the region of tumor, and does not spread substantially causing coagulation or tissue necrosis in healthy, normal tissues.

The Clotting Tissue Factor compound deficient or combinations thereof exercise in this way coagulatory and / or tissue destructive effects at a site of location of the disease or tumor and therefore have little or no coagulant or destructive effect of tissue in cells or normal, healthy tissues. Coagulation and / or tissue destruction therefore they are located at the site of disease or tumor and are not extend substantially or significantly to other vessels large or important blood or tissues. Therefore, in the methods of the invention cell function is maintained and Healthy tissues substantially without damage.

The `` Coagulation Tissue Factors deficient '' of the invention will generally be composed of Factor of Fabric that are at least one hundred times less active than the Factor Original fabric, full size, for example, when Test in a suitable phospholipid environment. Factor compounds of Fabric will still have activity, and are preferably described as being about a hundred times to about a million times less active than the original Fabric Factor of full size, for example, when tested in an environment suitable phospholipid.

Coagulation Tissue Factor Compounds deteriorated preferably will be at least about one thousand times less active than the original Fabric Factor, in size full; more preferably they will be at least ten thousand times less assets than the original full-size Fabric Factor; even more preferably they will be at least about one hundred thousand times less active than the full-size natural Tissue Factor, for example, when tested in a phospholipid environment suitable.

The `` at least approximately one hundred thousand times less active '' is not the minimum, and the tissue factor compounds they can be at least about five hundred thousand times or approximately one million times less active than the Factor of Original full-size fabric, for example, when tested on a suitable phospholipid environment.

Human Tissue Factor compounds will generally be preferred for human uses, but the use of other Tissue Factor species, including E. coli Tissue Factor, is not excluded. For ease of preparation, deficient coagulation tissue factor compounds will also preferably be prepared by recombinant expression, although this is not essential.

Tissue Factor can return coagulation deficient being deficient to bond with an area of phospholipid and / or deficient to be inserted into a membrane of phospholipid or lipid bilayer. Preferred examples are the `` Truncated Fabric Factors. '' As defined in the Patent USES. No. 5,504,064, in which the compounds are used for different purposes, `` truncated tissue factors '' they generally have an amino acid sequence that differs from the of the original Tissue Factor because enough amino acids from transmembrane that work to bind the Tissue Factor original with phospholipid membranes are missing in the protein of Truncated Tissue Factor, so that the Factor Factor protein Truncated tissue does not bind with phospholipid membranes.

Particular examples of Fabric Factors Truncated are Fabric Factor compounds that comprise approximately the first 219 contiguous amino acids of the Original Tissue Factor sequence, as exemplified further by a Fabric Factor compound consisting essentially in the amino acid sequence of Identification Sequence No. 1 (SEQ ID NO: 1). Although it is intended for use in different methods, U.S.A. No. 5,504,067 defines the Factors of Truncated tissue such as Tissue Factor proteins that have a amino acid sequence that begins at position 1 and ends near position 219 of the Tissue Factor sequence defined.

Fabric Factors can also be used deficient coagulation dimeric, including tissue factors homodimeric and heterodimeric. The dimers of tissue factors to Sample title are described in the present description as those consisting essentially of dimers of the sequence of amino acids of Identification Sequence No.3 (dimer H_ {6} -tTF_ {219} -cys-C '), Identification Sequence No.6 (dimer H_ {6} -tTF_ {220} -cys-C '), Sequence of Identification No.7 (dimer H_ {6} -tTF_ {221} -cys-C '), o Identification Sequence No.2 (dimer H_ {6} -N'-cys-tTF_ {219}). Chemically conjugated dimers, as described in detail later in this description, they are preferred for use in certain aspects of the present invention, although dimers recombinantly produced, in structure with linkers within a structure, they are also intended for use in particular realizations.

Coagulation Tissue Factor Compounds impaired for use with the present invention, they can also be Polymeric or multimeric Fabric Factors.

In certain embodiments, the Factor compound of Tissue will be a deficient mutant Tissue Factor in the ability to activate Factor VII. Although useful alone, uses more Preferred of these mutants will be in conjunction with the joint administration of a biologically effective amount of at least one Factor VIIa or an activator of Factor VIIa, such as when used with an amount of Factor VIIa sufficient to increase coagulation of tumor vasculature and necrosis of the tumor in the animal.

These mutants may be those that include a mutation in the amino acid region between approximately the position 157 and approximately position 167 of the Sequence of Identification No. 1. By way of example, but in no way limiting, are the mutants in which, within the sequence of Identification No.1, Trp at position 158 is changed to Arg; in where Being in position 162 is changed to Ala; where Gly in the position 164 is changed to Ala; or where Trp at position 158 is change to Arg and Ser at position 162 is changed to Ala. Examples defined of these mutants are those that consist essentially of The amino acid sequence of Identification Sequence No. 8 or Identification Sequence No.9.

Any of the Fabric Factor compounds of truncated, dimeric, multimeric and / or poor coagulation mutant can also be modified to increase longevity, life half or `` biological half-life '' of the Tissue Factor molecule. Several modifications of the structure of polypeptide in order to effect this change in the properties.

Particular examples of Fabric Factors modified to increase their biological half-life are the compounds of Tissue Factor that have been operatively linked, and preferably covalently linked to a carrier molecule, such as a protein carrier. The carriers preferably they are inert carriers, such as, by way of example only, an albumin or a globulin. They are also considered carriers no proteins such as polysaccharides and polymers synthetic

The operational union of a Factor construction from Tissue to an antibody or part thereof is a form currently Preferred Coagulation Factor Factor deficient with life increased biological mean. However, in the context of the first substance for use in anti-treatment strategies cancer disclosed in the present description, the Factor of Tissue will be bound to an antibody that shows no binding specific significant with a component of a tumor cell, tumor vasculature or tumor stroma. This is where the Tissue Factor compound is not bound to an antibody `` anti-tumor '', and when the compound of Factor of Resulting tissue is not a compound of `` Directed Tissue Factor to the tumor. ''

In these Factor conjugates Tissue-antibody, the compound of Tissue Factor it can be operatively linked to an IgG molecule called `` irrelevant specificity '', that is, one that has no affinity immuno-linker for a component of a tumor cell, tumor vasculature or tumor stroma. Factor compounds Fabric can also be operatively attached to an Fc part of a antibody, which has no specific objective function in the context of antibody specificity. Other constructions taken into account are those in which the compound of Factor of Tissue has been introduced into an IgG molecule instead of the domain C_ {H} 3.

Tissue Factor treatments surprisingly effective of the present invention can be combine advantageously with one or more other treatments. For example, treatment methods may further comprise administer to an animal or patient a biological amount or therapeutically effective of at least a second compound therapeutic, such as at least one of a second compound therapeutic selected from the group consisting of Factor VIIa, a Factor VIIa activator and at least a first substance or anticancer agent.

At least one first anticancer substance It can be a `` chemotherapeutic substance. '' As used in the In this description, the term `` chemotherapeutic substance '' is used to refer to a chemotherapeutic substance or drug classic used in the treatment of malignant tumors. This term is use for simplicity regardless of the fact that you can technically describe other compounds, including immunotoxins, as a chemotherapeutic substance because they exert an effect anticancer However, `` chemotherapeutic '' has come to have a different meaning in the art and is being used according to This standard meaning. `` Chemotherapeutic '' in the context of The present application, therefore, does not generally refer to immunotoxins, radiotherapeutic substances and the like, despite its operational overlap.

Several chemotherapeutic substances as example are indicated in Table II. The technicians in the field they will easily understand the appropriate uses and doses of the substances chemotherapeutic, although doses can be reduced satisfactorily when used in combination with this invention. A currently preferred chemotherapeutic substance is Etoposide A new class of drugs that can also be called `` chemotherapeutic substances '' are substances that induce apoptosis One or more of these drugs can also be used, including genes, vectors and antisense constructs, as appropriate suitable, together with the present invention.

Suitable anticancer substances include also specifically targeted toxic substances. For example anti-cancer antibodies and preferably constructs conjugated antibodies comprise an antibody that specifically binds to a component of a tumor cell, tumor vasculature or tumor stroma, where the antibody binds operationally or binds conjugates with at least a first cytotoxic substance or anti-cell or, for example, at least a first factor of coagulation.

By way of example only, the construction or target conjugate can be a construct or conjugate of antibody that specifically binds to a molecule of tumor cell surface; to a vasculature component tumor, such as E-selectin, P-selectin, VCAM-1, ICAM-1, endoglin or an integrin; to a component adsorbed or located in the vasculature or stroma, such as VEGF, FGF or TGFβ; to a component whose expression is naturally induced or artificially in the tumor environment, such as E-selectin, P-selectin or a MHC Class II antigen. The target substances that are not antibody include growth factors, such as VEGF and FGF; peptides containing the tripeptide R-G-D, that specifically bind to the vasculature of the tumor, and others target components such as annexins and ligands related.

Antibody constructs and conjugates they can be operatively linked to at least a first cytotoxic substance or otherwise anticellular. Can also be Operationally join at least a first coagulation factor. In conjunction with coagulants, they can also be used advantageously bispecific constructions (for example, using two regions of antibody binding), although covalent bonds are preferred Usually for use with toxins. One or more of the toxic substances or coagulants known in the art can be use in these `` immunotoxins '' or `` coaguligands '', and also they can use Fabric Factor or Fabric Factor derivatives as part of the coaguligands, where the coaguligand is the second `` anticancer substance ''.

The present invention therefore discloses also methods to treat an animal or patient that has a tumor vascularized, these methods generally include administering systemically to an animal one or more compounds of Tissue Factor of poor coagulation and one or more anticancer substances in a combined effective amount to coagulate the tumor vasculature and specifically induce tumor necrosis. The substance Anticancer can be a chemotherapeutic substance, such as exemplified by ethoposide, an antibody, or a construct of antibody or conjugate comprising an antibody that specifically it binds to a component of a tumor cell, a tumor vasculature or tumor stroma operatively attached to a cytotoxic substance or a coagulation factor.

Whether the anticancer substance is a chemotherapy or an antibody-based construct, the one or more anticancer substances can be administered to the animal simultaneously, for example, from a single composition or from Two or more different compositions. It is also considered the staggered or sequential administration of one or more compounds of Tissue Factor and one or more anticancer substances. The `` sequential administration '' requires that the Tissue Factor and the Anticancer substance administered to the animal at `` intervals of biologically effective time. '' For example, the compounds of Tissue Factor can be administered to the animal in a moment biologically effective before the anticancer substance or substances, or the anticancer substance or substances can be administered to the animal at a biologically effective time before the compound or Tissue Factor compounds. When you first administer a Fabric Factor compound, will usually be given in a moment biologically effective enough to allow the compound of the Tissue Factor is preferentially located within the vasculature of the tumor before the administration of the substance or anticancer substances.

The present invention also includes methods for use at least one Factor VIIa or a Factor VIIa activator to increase the effectiveness of one or more of the Factor compounds Poor coagulation tissue that defines primary therapy. These methods generally comprise also administering to an animal. or patient a therapeutically effective amount of Factor VIIa or a Factor VIIa activator.

In these embodiments, it will generally be preferred the use of Factor VIIa alone. The Factor VIIa employed may consist essentially in the amino acid sequence of the Sequence of Identification No.14.

Again, Factor VIIa or the activator of Factor VIIa can be administered to the animal simultaneously with the Coagulation Factor Factor deficient coagulation. As such, Factor VIIa can be administered to the animal or patient in a preformed complex of Tissue Factor-Factor VIIa. In certain embodiments, the Factor complex of Tissue-Factor VIIa will be an equimolar complex.

In addition, the Fabric Factor compound of poor coagulation and Factor VIIa compound can be administer to the animal using staggered or sequential administration. The first administration of the Fabric Factor compound generally it will be preferred and it will be preferable to administer it to the animal in a biologically effective time before the Factor compound VIIa. This prior effective administration of the Factor compound Fabric will generally be at a biologically effective time to allow the Fabric Factor compound to preferentially be locate inside the vasculature of the tumor before the administration of the Factor VIIa compound.

These methods, therefore, can be described as methods to promote coagulation in the vasculature of the tumor of an animal or patient that has a vascularized tumor, which comprises systemically providing the animal or patient with a Composite of Deficient Coagulation Tissue Factor and Factor VIIa or a Factor VIIa activator in a combined amount enough to preferentially or specifically promote the coagulation in the vasculature of the tumor.

The subject animal is preferably will provide coagulation tissue factor compound deficient at a time prior to providing Factor VIIa, so that the time interval prior to administration of Factor VIIa is effective for the compound of Factor of Preferentially woven or specifically located within the Vasculature of the tumor

Other methods are described as the methods for treat an animal that has a vascularized tumor, which comprise systemic administration to the animal of a compound of Factor of Poor coagulation tissue and Factor VIIa in an amount combined effective to promote coagulation in the vasculature of the tumor and specifically cause tumor necrosis. The Pre-administration of the Tissue Factor is generally preferred by so that the Fabric Factor compound is preferentially locate inside the vasculature of the tumor and form a reserve for the subsequent combination of Factor VIIa.

All these combined Factor treatments VIIa can be used with any Fabric Factor compound of poor coagulation, such as truncated, dimeric, and / or mutant and / or those with increased half-life. These methods are particularly useful for combination with Fabric Factor compounds that are poor in their ability to activate Factor VII.

The combined treatment methods of the invention also encompass triple combinations that use one or more Coagulation Factor Factor compounds deficient, one or more Anticancer substances and Factor VIIa or a Factor activator VIIa.

The present invention further discloses novel compositions in the form of compositions comprising one or more compounds of deficient coagulation tissue factor that have been modified to increase their half-life, different from others in which the modification consists of joining the compound of Tissue Factor to an antibody that binds to a component of a tumor cell, tumor vasculature or tumor stroma.

The `` Life Tissue Factor compounds increased mean '' encompasses all Tissue Factor compounds of poor coagulation described above, such as truncated, dimeric, polymeric, and / or mutant tissue factors which have been modified by increasing their half-life.

The half-life tissue factor compounds augmented preferably comprise a compound of Factor of Poor coagulation tissue that is operatively linked, by example, covalently bound, to a carrier molecule. The protein carriers are currently preferred, as exemplified by albumin or globulins, although they are also They consider non-protein carriers.

A class of Fabric Factor compounds of poor coagulation of increased half-life are those that are operably linked to an antibody or part thereof, such as an IgG molecule or to an Fc part of an antibody. Too tissue factors introduced into one part are considered contiguous of an IgG molecule, for example, in the place of domain C_ {H} 3.

The invention also discloses a series of novel therapeutic equipment for use in conjunction with the described methods. These equipment will comprise, preferably in convenient container elements, at least a first compound of Deficient Coagulation Tissue Factor in combination with at least a first anticancer substance.

Coagulation Tissue Factor Compounds deficient may be one or more of the tissue factors of poor coagulation described in the present description, such such as Truncated, Dimeric, Polymeric, and / or Tissue Factors mutants, which include mutant tissue factors deficient in the ability to activate Factor VII. When mutants are used of activation of Factor VII in the equipment or `` kit '', this optionally it can also comprise a quantity biologically Effective Factor VIIa.

The term `` anticancer substance '' is used as It has been described above and covers chemotherapeutic substances, such as etoposide; and anticancer substances based on antibodies, such as antibody conjugates comprising a antibody that specifically binds to a component of a tumor cell, tumor vasculature or tumor stroma operatively bound to a cytotoxic substance or a coagulation factor, which includes a Tissue Factor or a Tissue Factor derivative.

Other therapeutic equipment of the invention generally comprise, preferably in container elements convenient, a mutant Tissue Factor compound that is deficient in its ability to activate Factor VII in combination with Factor VIIa. Previously, it had been thought that mutants of this category lacked activity so they could not be used therapeutically to induce coagulation, but only to act as a tissue type antagonist of wild type e inhibit clotting Only the combination of the Factor of Truncated fabric substantially active with Factor VIIa has been previously proposed, in relation to the treatment of disorders blood.

The present invention thus discloses the novel combination of a mutant Tissue Factor compound which is more significantly impaired in its capacity coagulant than the Truncated Tissue Factor, preferably in by virtue of being deficient in the ability to activate Factor VII, together with Factor VIIa. Factor VIIa will become `` Factor VIIa exogenous '' after administration to an animal. This teams, therefore, they preferably comprise, in a container element convenient, a biologically effective amount of a compound of Mutant tissue factor deficient in its ability to activate the Factor VII in combination with a biologically effective amount of at least one Factor VIIa or an activator of Factor VIIa. The Factor VIIa activators can be replaced by Factor VIIa in these equipments, or they can be used in addition to Factor VIIa. Supplementary substances can also be added.

Tissue Factor mutants for use in these teams are exemplified by those that include a mutation in the amino acid region between approximately position 157 and approximately position 167 of the Identification Sequence No.1. This is exemplified by mutants in which, within of Identification Sequence No.1, the Trp at position 158 change to Arg; where Being in position 162 changes to Ala; where Gly at position 164 changes to Ala; or where Trp in position 158 changes to Arg and Ser at position 162 changes to Ala. Others examples are the mutant tissue factors that consist essentially in the amino acid sequence of the Sequence of Identification No.8 or Sequence of Identification No.9.

The combined treatment equipment that they comprise, preferably in a convenient container element, at least the first coagulation tissue factor compound deficient, at least a first anticancer substance and Factor VIIa or a Factor VIIa activator, are also disclosed by the invention.

Brief description of the drawings

The following drawings are part of the present descriptive report and are included to demonstrate additionally certain aspects of the present invention. The invention can be better understood by referring to one or more of these drawings in combination with the detailed description of the specific embodiments presented in this patent.

Figure 1: Plasma coagulation induction Through full-size Fabric Factor. The blood (A) is sample in contact with the cell membrane (B).

Figure 2: The domain structure of the Factor of Tissue. The extracellular domain (A; amino acids is represented 1-219), and the cell membrane (B). The Dominion NH2 (10) is represented as cross hatching, the region of Factor VII / VIIa link (20) is represented as shaded. The transmembrane domain (40) begins at amino acid 220 (30) and It encompasses the cell membrane. The transmembrane domain of the Factor Tissue is suppressed or becomes non-functional otherwise to generate a functional (FTt) of the present invention. In certain compositions of (FTt) the NH2 domain can also be deleted or back not functional.

Figure 3: Model for coagulation induced by Truncated Tumor Vasculature Tissue Factor. The blood (A) is represents in contact with the cell membrane (B) of the endothelium vascular tumor (C). The prothrombotic tumor endothelium has factor IX, X (shown) or TFPI / Xa plus serine phosphatidyl (PS -) on its surface, which links tTF-VII or tTF-VIIa, leading to coagulation.

Figures 4A and 4B. Figure 4A: Induction of coagulation by the truncated Tissue Factor linked to the cell. I know incubated A20 cells (10 5 cells, 100 µl) with antibodies (0.33 \ mug) and Truncated Tissue Factor (0.17 \ mug) during a hour at 4 ° C. Calcium chloride (12.5 mM) and plasma were added mouse cited to the cells and the time was recorded for they will form the first strands of fibrin (coagulation time, seconds; horizontal axis). The sample number is shown on the axis vertical. Sample 1 does not include added antibodies or Factor of Truncated tissue (control), sample 2 includes antibody B21-2 / 10H10, Sample 3 includes Fabric Factor truncated, sample 4 includes B21-2 / OX7 antibody plus Truncated Tissue Factor, sample 5 includes antibody CAMPATH-2 / 10H10 plus Truncated Fabric Factor, the Sample 6 includes 10H10 F (ab ') 2 antibody plus Factor Truncated tissue, sample 7 includes 10H10 Fab 'antibody plus Truncated Tissue Factor, sample 8 includes antibody B21-2 F (ab ') 2 plus Tissue Factor truncated, sample 9 includes B21-2 Fab 'antibody plus Truncated Tissue Factor, sample 10 includes antibody B21-2 / 10H10 plus Truncated Fabric Factor. Figure 4B: Relationship between the number of Tissue Factor molecules truncated ligands and plasma coagulation time. They incubated A20 cells (10 5 cells, 100 µl) with concentrations variants of B21-2 / 10H10 plus an excess of Factor of Truncated tissue for one hour at 4 ° C in the presence of azide sodium and then washed, heated to 37 ° C. Chloride was added of calcium (12.5mM) and citrated mouse plasma (a different batch of from A) to the cells and the time for the formation of the cells was recorded first strands of fibrin (coagulation time, seconds; axis vertical). The number of bound truncated Tissue Factor molecules to cells (?) was determined in a parallel study with 125 I-tTF (log scale; horizontal axis). The values represent the average of the three measures, with deviation standard.

Figure 5: Mouse plasma coagulation by tTF_ {219}, H_ {6} -N'-cys-tTF_ {219} Y H_ {6} -tTF_ {219} -cys-C ' associated with cells. A20 lymphoma cells were treated (I-A d positive) at room temperature with the bispecific `` capture '' antibody, B21-2 / 10H10,  recognizing both I-A d and the Factor of Truncated fabric. The cells were washed and two were added different preparations of Truncated Tissue Factor 219 [tTF_ {219} (\ medcirc) standard and tTF_ {219} (\ blacktriangle)], H_ {6} -N'-cys-tTF_ {219} (\ Box) or H_ {6} -tTF_ {219} -cys-C '(\ newmoon) at a range of concentrations of Truncated Tissue Factor (concentration, M; horizontal axis). The cells were washed and heated at 37 ° C. Calcium was added and citrated mouse plasma and recorded the time to form the first strands of fibrin (coagulation time, seconds; axis vertical).

Figure 6: Mouse plasma coagulation by H_ {6} -tTF_ {220} -cys-C '  and tTF_ {220} -cys-C 'associated with the cell. A20 lymphoma cells were treated (I-A d positive) at room temperature with the bispecific `` capture '' antibody, B21-2 / 10H10, recognizing both I-A d and the Factor of Truncated fabric. The cells were washed and tTF 219 was added standard (\ blacksquare), H_ {6} -tTF_ {220} -cys-C '  (\ medcirc) and tTF_ {220} -cys-C ' (?) to a range of concentrations (concentration, M; axis horizontal). The cells were washed and heated at 37 ° C. I know added calcium and citrated mouse plasma and the time was recorded for that the first strands of fibrin were formed (time of coagulation, seconds; vertical axis).

Figure 7: Mouse plasma coagulation by H_ {6} -tTF_ {221} -cys-C ',  tTF_ {221} -cys-C 'and dimer of H_ {6} -tTF_ {221} -cys-C ' associated with cells. A20 lymphoma cells were treated (I-A d positive) at room temperature with bispecific `` capture '' antibody, B21-2 / 10H10,  recognizing both I-A d and Tissue Factor truncated. Cells were washed and standard tTF 219 was added (\ medcirc), H_ {6} - tTF_ {221} -cys-C '(\ newmoon), tTF_ {221} -cys-C '(\ Box), or dimer of H_ {6} -tTF_ {221} -cys-C ' (iangle) at a range of concentrations (concentration, M; horizontal axis). The cells were washed and heated at 37 ° C. I know added calcium and citrated mouse plasma and the time was recorded to form the first strands of fibrin (time of coagulation, seconds; vertical axis).

Figure 8: Mouse plasma coagulation by H_ {6} -N'-cys-tTF_ {219}  and dimer of H_ {6} -N'-cys-tTF_ {219} associated with the cell. A20 lymphoma cells were treated (I-A d positive) at room temperature with the bispecific `` capture '' antibody, B21-2 / 10H10, recognizing both the I-A d and the Factor of Truncated fabric. The cells were washed and tTF 219 was added standard (\ medcirc), H_ {6} -N'-cys- tTF_ {219} (\ blacksquare) and dimer of H_ {6} -N'-cys-tTF_ {219} (?) in a range of concentrations (concentration, M; axis horizontal). The cells were washed and heated at 37 ° C. I know added calcium and citrated mouse plasma and the time was recorded for the first strands of fibrin to form (time of coagulation, seconds; vertical axis).

Figure 9: Thrombosis of vessels in large tumors C1300 Muγ using tTF_21. Nu / Nu mice that had Large subcutaneous C1300 Mu [gamma] tumors (> 1000 cm 3) are injected intravenously with 16-20 µg of tTF_ {219}. Twenty four hours later, the mice were anesthetized, bled and removed the tumors and organs Paraffinic tissue sections were evaluated for Determine the presence of vessels with thrombosis. The amount of glasses with thrombosis and open vessels in tumor sections were counted. The percentage of tumor vessels with thrombosis is shown on the axis vertical. The shaded bar represents mice injected with tTF_21, the open bar represents mice injected with PBS (`` phosphate regulated saline solution '').

Figure 10: Thrombosis of vessels in large tumors C1300 using tTF_ {219}. Nu / Nu mice that had C1300 tumors large subcutaneous (> 1000 mm3) were injected intravenously with 16-20 µg of tTF_ {219}. Twenty four hours later, the mice were anesthetized, they they bled and their tumors and organs were removed. I know evaluated paraffinic sections of the tissues to determine the presence of vessels with thrombosis. The number of vessels with thrombosis and open vessels in tumor sections were counted. The percentage of tumor vessels with thrombosis is shown on the vertical axis. The shaded bar represents mice injected with tTF_21, the open bar represents mice injected with saline phosphate regulated.

Figure 11: Thrombosis of vessels in large tumors 3LL using tTF_ {219}. C57BL / 6 mice that had 3LL tumors large subcutaneous (> 800 mm3) were injected intravenously with 16-20 µg of tTF_ {219}. Twenty four hours later, the mice were anesthetized, they they bled and their tumors and organs were removed. I know evaluated paraffinic sections of the tissues to determine the presence of vessels with thrombosis. The number of vessels with thrombosis and open vessels in tumor sections were counted. The percentage of tumor vessels with thrombosis is shown on the vertical axis. The shaded bar represents mice injected with tTF_21, the open bar represents mice injected with saline phosphate regulated.

Figures 12A and 12B: Growth inhibition of C1300 Muγ tumors in mice by tTF21. Figure 12A: To mice with C1300 tumors (Muγ) from 0.8 to 1.0 centimeters in diameter were given two injections coaguligand intravenous B21-2 / 10H10-tTF (\ newmoon) separated by six days (arrows). To the mice in the group of control were given equivalent doses of Tissue Factor truncated alone (\ Box), CAMPATH-2 / 10H10 plus tTF (Δ) or phosphate buffered saline solution (cir). The mice that received B21-2 / OX7 and tTF had Tumor responses similar to animals that received tTF alone. The administration of B21-2 / 10H10 did not affect just tumor growth. Each group contained 12 to 27 mice. The dots represent the average tumor volume per group (± SEM). The average tumor volume (cm3) is shown in the vertical axis, days after the first treatment is shown in the horizontal axis Figure 12B: A Nu / nu mice that had tumors C1300 Small subcutaneous Mu [gamma] (350 mm3) were injected intravenously with 16-20 micrograms of tTF_ {219} (\ blacksquare) or phosphate regulated saline (\ medcirc). The treatment was repeated a week later. The tumors were measured daily and the volumes of the tumor (+ a standard deviation). The number of mice per group of Treatment was 8-10. The average volume of tumor (cm3) is shown on the vertical axis, days after First treatment is shown on the horizontal axis.

Figure 13: Inhibition of tumor growth H460 in mice by tTF 219. To Nu / nu mice that had Small subcutaneous H460 tumors (350 mm3) were injected intravenously with 16-20 micrograms of tTF_ {219} (\ blacksquare) or phosphate regulated saline (\ medcirc). The treatment was repeated a week later. The Injection time is designated by arrows. Tumors are measured daily and tumor volumes were calculated (+ a standard deviation). The number of mice per group of Treatment was 8-10. The average volume of tumor (cm3) is shown on the vertical axis, days later the First treatment is shown on the horizontal axis.

Figure 14: Inhibition of tumor growth HT29 in mice by tTF 219. To Nu / nu mice that had large subcutaneous HT29 tumors (1200 mm3) were injected intravenously with 16 micrograms or 64 micrograms of tTF 219 (Box) or phosphate regulated saline solution (moon). To the tumors were measured daily (days after injection; axis horizontal), and tumor volumes were calculated (+ one standard deviation (tumor volume, mm3; vertical axis). The number of mice per treatment group was 3-4.

Figure 15: Mouse plasma coagulation through IgG-H6 -N'-cys- tTF 219 associated with cells. A20 lymphoma cells were treated (I-A d positive) with bispecific antibody of `` capture '', B21-2 / 10H10, recognizing both I-A d as tTF 219. They were added IgG-H6 -N'-cys- tTF_ {219} (Δ), H_ {6} -N'-cys-tTF_ {219} (\ blacksquare) or tTF_ {219} (\ medcirc) at a range of concentrations at room temperature (concentration, M; axis horizontal). The cells were washed and heated at 37 ° C. I know added calcium and citrated mouse plasma and the time was recorded for  that the first strands of fibrin were formed (time of coagulation, seconds; vertical axis).

Figure 16: Mouse plasma coagulation by IgG-H_ {6} -N'-cys-tTF_ {219}  and IgG-H6 -tTF_ {219} -cys-C ' associated with cells. Conjugates of immunoglobulin-tTF binding B21-2 IgG (with respect to I-A d) a H_ {6} -N'-cys-tTF_ {219} (\ blacktriangle) or H_ {6} -tTF_ {219} -cys-C ' (\ blacksquare). Conjugates were added to a range of A20 lymphoma cell concentrations (I-A d positive) at room temperature, and were compared with tTF 219 (?) (concentration, M; horizontal axis). The cells are washed and heated at 37 ° C. Calcium and mouse plasma were added cited The time (seconds) was recorded for the formation of the first strands of fibrin. The vertical axis shows the time of coagulation as a percentage of control.

Figure 17: Conversion from Factor X to Factor Xa through IgG-H_ {6} -N'-cys-tTF_ {219} associated with cells and Fab'-H_ {6} -N'-cys- tTF 219, measured by a chromogen test. They were treated A20 cells (I-A d positive) with the antibody Bispecific `` capture '', B21-2 / 10H10, recognizing both I-A d and tTF, it was added with IgG-H6 -N'-cys- tTF_ {219} (\ medcirc) or Fab'-H6-N'-cys-tTF_ {219} (\ bigtriangleup), which were added to a range of concentrations at room temperature. Were also added B21-2 / 10H10 more H_ {6} -N'-cys-tTF_ {219} (X) and Mac51 / 10H10 more H_ {6} -N'-cys-tTF_ {219} (control, \ blacksquare) (concentration, M; horizontal axis). The cells were washed and heated at 37 ° C. Calcium was added and `` proplex t '' (the proplex t contains factors II, VII, IX and X). The Xa production was measured by adding the substrate that releases chromophore, S-2765, and measuring the optical density at 409 nm (OD 409 nm; vertical axis).

Figure 18: Inhibition of tumor growth C1300 Muγ in mice by conjugate of immunoglobulin-tTF. To Nu / nu mice that had Small subcutaneous C1300 Mu γ tumors (300 mm 3) were given injected intravenously with 16-20 micrograms of tTF_ 219 complexed with bispecific `` carrier '' antibody OX7 Fab '/ 10H10 Fab (Δ). Other mice received tTF 219 alone  (\ blacksquare), or diluent (PBS, \ medcirc). The treatment is repeated a week later. Daily treatments are designated by arrows Tumors were measured daily and calculated. tumor volumes (plus a standard deviation). The number of mice per treatment group was 7-10. The average tumor volume (cm3) is sample on the vertical axis, days after the first treatment is shows on the horizontal axis.

Figure 19: Increase in antitumor activity of the immunoglobulin-tTF by the ethoposide. TO SCID mice that had subcutaneous L540 human Hodgkin tumors a single intravenous injection of a complex of tTF_21 and the bispecific `` carrier '' Mac51 antibody Fab '/ 10H10 Fab' (\ blacksquare). Other mice received 480 Etoposide intraperitoneally two days later, one day after and on the day of treatment with the conjugate of immunoglobulin-tTF (black). Others mice received etoposide alone (?) or diluent (phosphate regulated saline, Δ). Tumors are measured daily and tumor volumes were calculated (+ a standard deviation). The average tumor volume (cm3) is shown on the vertical axis, days after Treatment is shown on the horizontal axis.

Figure 20: Increased plasma coagulation by Factor VIIa. A20 lymphoma cells were treated (I-A d positive) at room temperature with the bispecific `` capture '' antibody, B21-2 / 10H10,  recognizing both I-A d and tTF. The cells washed and added tTF 219 alone (?) or tTF_21 with Factor VIIa in a range of concentrations of Factor VIIa, as follows: 0.1 nM (\ blacksquare); 0.3 nM (\ newmoon); 0.9 nM (Δ); 2.7 nM (iangle); and 13.5 nM (+) (concentration, M; horizontal axis). The cells were washed and heated to 37 ° C. Calcium and citrated mouse plasma were added and the time for the first strands of fibrin (coagulation time, seconds; vertical axis).

Figure 21: Weak mouse plasma coagulation by tTF 219 (W158R) and tTF 219 (G164A) mutants associated with cells. A20 lymphoma cells were treated (I-A d positive) at room temperature with the bispecific `` capture '' antibody, B21-2 / 10H10, recognizing both I-A d and tTF. The cells washed and added tTF_21 (\ medcirc), tTF_ {219} (W158R) (\ newmoon) or tTF_ {219} (G164A) (\ Box) at a range of concentrations (concentration, M; horizontal axis). The cells are washed and heated at 37 ° C. Calcium and mouse plasma were added cited and recorded the time for the first to form fibrin strands (coagulation time, seconds; axis vertical).

Figure 22: Restoration of inductive activity of coagulation of the tTF 219 mutant (G164A) and (W158R) by Factor VIIa. A20 lymphoma cells were treated (I-A d positive) at room temperature with the bispecific `` capture '' antibody, B21-2 / 10H10, recognizing both I-A d and tTF. The cells washed and not treated (Δ) or treated with: tTF_ {219} (\ medcirc); tTF_ {219} (G164A) (\ blacksquare) or tTF_ {219} (W158R) (\ newmoon); each with the addition of Factor VIIa at a range of concentrations (concentration, nM; axis horizontal). The cells were washed and heated at 37 ° C. I know added calcium and citrated mouse plasma and the time was recorded to form the first strands of fibrin (time of coagulation, seconds; vertical axis).

Figure 23: Complex antitumor activity tTF219: VIIa and tTF219 (G164A): VIIa in mice that have carcinomas HT29 human colorectals. From left to right, the bars represent: saline solution (1); tTF (2); Factor VIIa (3); tTF more Factor VIIa (4); G164A (5); and G164A plus Factor VIIa (6). The axis vertical shows the average percentage of necrosis in tumors examined.

Sequence Summary

       \ dotable {\ tabskip \ tabcolsep # \ hfil \ + # \ hfil \ tabskip0ptplus1fil \ dddarstrut \ cr} {
 SEQ ID NO: 1 \ + Amino acid sequence of tTF 219 \ cr SEQ ID
NO: 2 \ + Amino Acid Sequence of
H 6 -N'-cys-TF 219
 SEQ ID NO: 3 \ + Amino Acid Sequence of
H 6 -tTF 219 -cys-C '\ cr
 SEQ ID NO: 4 \ + Amino Acid Sequence of
N'-cys-tTF 219 \ cr SEQ ID NO: 5
\ + Amino Acid Sequence of
tTF 219 -cys-C '\ cr SEQ ID NO: 6
\ + Amino Acid Sequence of
H 6 -tTF 220 -cys-C '\ cr
 SEQ ID NO: 7 \ + Amino Acid Sequence of
H 6 -tTF 221 -cys-C '\ cr
 SEQ ID NO: 8 \ + Amino Acid Sequence of tTF 219 (W 158
R) cr SEQ ID NO: 9 \ + Amino acid sequence of tTF 219 (G
164 A) \ cr SEQ ID NO: 10 \ + cDNA sequence for tTF \ cr SEQ ID
NO: 11 \ + Complete genomic sequence of Tissue Factor \ cr SEQ
ID NO: 12 \ + Tissue Factor amino acid sequence \ cr SEQ
ID NO: 13 \ + Factor VII DNA \ cr SEQ ID NO: 14 \ + Amino Acids
Factor VII \ cr SEQ ID NO: 15 \ + 5 'primer for amplification of
tTF \ cr SEQ ID NO: 16 \ + 3 'primer for amplification of tTF \ cr
SEQ ID NO: 17 \ + 5 'GlytTF primer DNA amplification primer
Complementary \ cr SEQ ID NO: 18 \ + 5 'primer for preparation of
tTF and the 5 'half of the DNA linker \ cr SEQ ID NO: 19 \ + 3' primer
for the preparation of tTF and the 5 'half of the DNA linker? cr SEQ
ID NO: 20 \ + 5 'primer for the preparation of the 3' half of the AND
linker and DNA tTF \ cr SEQ ID NO: 21 \ + 3 'primer for the
Preparation of the 3 'half of the linker DNA and the tTF \ cr DNA SEQ ID
NO: 22 \ + 5 'primer for Cys [tTF] linker [tTF]
Construction \ cr SEQ ID NO: 23 \ + 3 'primer for Cys [tTF]
linker [tTF] construction \ cr SEQ ID NO: 24 \ + 5 'primer
for [tTF] linker [tTF] cys \ cr SEQ ID NO: 25 \ + 3 'primer for
[tTF] linker [tTF] cys \ cr SEQ ID NO: 26 \ + Primer for the
formation of [tTF] G164A \ cr SEQ ID NO: 27 \ hskip1cm \ +
Primer for [tTF] W158R
S162A \ cr}
    

Description of the illustrative embodiments

Solid tumors and carcinoma are more than 90 percent of all cancers in the human (Shockley and collaborators, 1991). The therapeutic uses of antibodies Monoclonal and immunotoxins have been investigated in therapy of lymphomas and leukemias (Lowder et al., 1987; Vitetta and collaborators, 1991), but they have been disappointingly ineffective in clinical trials against carcinomas and other solid tumors (Byers and Baldwin, 1988; Abrams and Oldham, 1985).

A main reason for the inefficiency of antibody-based treatments is that macromolecules don't easily transport to solid tumors (Sands, 1988; Epenetos et al., 1986). Even when these molecules reach the tumor mass, fail to distribute evenly due to the presence of strong junctions between tumor cells (Dvorak and collaborators, 1991), fibrous stroma (Baxter et al., 1991), interstitial pressure gradients (Jain, 1990) and barriers of binding site (Juweid et al., 1992).

To develop new strategies to try solid tumors, the methods that involve targeting the vasculature of the tumor, instead of the same tumor cells, They offer different advantages. Inducing a blockage of the flow of blood through the tumor, for example, through the formation of specific fibrin of the tumor vasculature, would interfere with the processes of influx and ebb in a location site of the tumor, resulting in an antitumor effect. Stop the blood supply to a tumor can be carried out through of changing the procoagulant, fibronilitic balance in the vessels associated with the tumor in favor of coagulant processes through specific exposure to coagulant substances. In accordance with the above, have been generated and used in the specific administration of a coagulant to the tumor environment, constructions of coagulant antibodies and bispecific antibodies (WO 96/01653).

However, the requirement for specificity, Although it is not as strict as in immunotoxins, it is still important. To achieve specificity, it has generally been believed that an effector molecule, either a toxin or a coagulant, it needs to be conjugated or functionally associated with a molecule objective, such as an antibody or other ligand with specificity for the tumor environment. These target entities can be target the same tumor cells, although it is now believed to be it is preferable to use target molecules directed against components of the vasculature of the tumor or stroma of the tumor. They have been identified several suitable target molecules that are specific or preferentially expressed, localized, adsorbed or induced in the cells or in the environment of the tumor vasculature and / or stroma

Although the methods that target the vasculature and stroma of the tumor can be quite effective, it will recognize that the practice of this objective methodology still requires certain knowledge and requires the preparation of convenient conjugates or coordinated molecular complexes. By For example, to direct a coagulant to the vasculature of the tumor, must identify a suitable vascular antigen, prepare a antibody or ligand that binds to the target antigen, choose a suitable coagulant, bind the coagulant with the antibody or ligand or otherwise form a functional association of the two components, and conduct location protocols using doses that do not result in a significant incorrect address of the substance. Although these methods can be easily practiced and successfully, it can be seen that the advantages would be the result of methodology development that includes fewer preparatory stages and therefore it could be done in a more effective way in cost.

The present invention discloses compositions to use in these new methods to effect coagulation of specific blood, as exemplified by specific coagulation of tumor, without the need for target molecules, such as antibodies This is achieved by administering compositions that comprise deficient coagulant tissue factor, which has been discovered that specifically promotes coagulation in the vasculature of the tumor, despite the fact that it lacks any target component of recognized tumor. The present invention gives know that these coagulation tissue factor compositions impaired can be administered alone, as a conjugate of Factor of Fabric with improved half-life, in combination with chemotherapy conventional, in combination with immunotoxins or coaguligands objective, in combination with Factor VIIa (FVIIa) or activators of FVIIa factor or any of the above combinations.

A. Fabric Factor

Tissue Factor (FT) is the receptor of most important initiation for thrombogenic cascades (blood coagulation) (Davie, et al., 1991). The Factor of Tissue is a single chain, 263 membrane glycoprotein amino acids (SEQ ID NO: 12), and its primary sequence has structural similarity with the chemokine receptor family (Edgington et al., 1991). The Tissue Factor is a transmembrane cell surface receptor and functions as the receptor and cofactor for Factor VIIa. Tissue Factor links Factor VIIa to form a proteolytically active complex in the cell surface (Ruf and Edgington, 1991b, 1994; Ruf and collaborators, 1991, 1992a, 1992b). This rapidly active complex Serine protease zymogens Factors IX and X by limited proteolysis, leading to thrombin formation and, finally, to a blood clot (figure 21).

Thus, the Tissue Factor is an activator. of the extrinsic path of blood coagulation and is not in direct contact with blood under physiologically conditions normal (Osterud et al., 1986; Nemerson, 1988; Broze, 1992; Ruf and Edgington, 1994). In vascular damage or activation by certain cytokines or endotoxin, however, the Factor of Tissue will be exposed to blood, either by cells (sub) endothelial (Weiss et al., 1989) or through certain blood cells (Warr et al., 1990). The tissue factor will be complexed with Factor VIIa, which under normal conditions circulates at low concentrations in the blood (Wildgoose and collaborators, 1992), and the TF / Factor VIIa complex will begin the coagulation cascade through the activation of Factor X in Xa factor. The waterfall will eventually result in the formation of fibrin (figure 1). For this sequence of events to occur, the TF complex: VIIa has to be associated with a surface of phospholipid on which the initiation complexes of the Coagulation with Factors IX or X can be assembled (Ruf and Edgington, 1991a; Ruf et al 1992c; Paborsky and collaborators 1991; Bach et al 1986; Krishnaswamy and collaborators 1992; Ten Cate et al 1993).

A limited number of cells constitutively express the tissue factor. Lung and system tissues Central nervous contain high levels of Factor activity Tissue, finding Tissue Factor in bronchial mucosa and cells alveolar epithelials in the lung and glial cells and astrocytes in the nervous system. Tissue Factor Expression It has also occurred in cardiac myocytes, renal glomeruli, and in certain epithelial or mucosal tissues of the intestine, the Bladder and respiratory tract. In this way you can see that the Tissue Factor is generally expressed constitutively as tissue barriers between body tissues and the environment external (Drake et al., 1989; Ruf and Edgington, 1994).

Tissue Factor is also present in tissue boundaries between organs, such as in capsules of organs of the liver, spleen and kidney, and is also present in the adventitia of arteries and veins. Tissue Factor Expression in this way it allows the Tissue Factor to work to stop internal bleeding Therefore it is important to note that the Tissue Factor is absent in the joints and muscle skeletal hemophiliacs, which are the first sites of hemorrhage in these patients.

Tissue Factor is typically not expressed in no degree in blood cells or on the surface of cells endothelials that form the vasculature under normal conditions, but its expression by (sub) endothelial cells and monocytes Within the vasculature it can be induced by infectious agents. Monocytes, for example, are induced to express Factor of Tissue using cytokines and T cells. The expression of Factor Tissue in the vasculature will typically result in coagulation disseminated intravascular or localized clot initiation blood or thrombogenesis In this context, it is important to note that the Fabric Factor must be available at all sites of the body where coagulation would be necessary after damage, infection of the tissue or other aggressions. Therefore, the Factor of Fabric must be equally available in all those tissue sites and should not generally be reserved within a particular localized area of the body.

Certain studies have led to delineation of a relationship between the Tissue Factor and the development of Neoplastic phenotype in certain types of tumors (Ruff and Edgington, 1994). In fact, increasing levels of Factor have been released of Tissue as an indicator of metastatic potential prognosis of malignant melanoma (Mueller, et al., 1992). Has reasoned that a generalized activation of the coagulation cascade could damage the vasculature leading to the access of tumor cells or vesicles derived from tumor cells to the circulation general, allowing these tumor cells to mature and cause metastatic tumor growth.

Regardless of the underlying mechanism, the studies described above have led to Edgington and collaborators to propose the use of antibodies directed against the Tissue Factor in cancer treatment (WO 94/05328). These authors, therefore, have proposed that antibodies with binding affinity for Tissue Factor have therapeutic utility in cancer treatment, particularly in relation to patients believed to be at risk of developing metastatic tumors This attempt has led to the development of hybridomas that produce monoclonal antibodies that react with Human tissue factor (U.S. Patent No. 5,223,427).

In addition to the use in the treatment against cancer, tissue factor antibodies have also been proposed for use in the inhibition of excessive coagulation, the which can also be used in relation to shock treatment septic and in moderating inflammatory responses (Morrissey and collaborators, 1988; U.S.A. number 5,223,427), or in the myocardial infarction treatment, where antibodies are used as tissue factor antagonists (U.S. Patent No. 5,589,173). The combined use of Tissue anti-factor antibody and other thrombolytic substances to dissolve occluding thrombi will particularly described in U.S. Pat. No. 5,589,173. A specific method to use these antibodies is in the inhibition of coagulation in the extracorporeal circulation procedure in which blood is withdrawn from a patient during a surgical procedure, such as a diversion procedure cardiopulmonary (U.S. Patent No. 5,437,864).

How it develops more fully later (Section B), the Human Tissue Factor has been cloned and is available for some time (Morrissey et al., 1987; Edgington et al., 1991; U.S.A. No. 5,110,730). In certain previous studies, the same protein currently identified as Human Tissue Factor is known as heavy chain protein of human tissue factor or chain Heavy tissue factor. The gene encodes a precursor of polypeptide of a size of 295 amino acids, which includes a front peptide with alternative dissociation sites, which known to drive the formation of a 263 amino acid protein from size. Recombinant expression of Human Tissue Factor in CHO cells have been reported to lead to the production of Factor Woven at a level that is described as being one of the levels of highest expression disclosed for a recipient of recombinant transmembrane after production in cells of mammals (Rehemtulla et al., 1991).

A recombinant form of Factor has been constructed Tissue that contains only the cell surface or domain extracellular (Stone, et al., 1995) and lacks the transmembrane and cytoplasmic regions of Tissue Factor. This `` Truncated '' Tissue Factor (tTF) is 219 in size amino acids and is a soluble protein with approximately 10 5 times less X-activating factor of relative activity to the original Transmembrane Tissue Factor in an environment of Suitable phospholipid membrane (Ruf, et al., 1991b). Is difference in activity is due to the fact that the TF complex: VIIa league and activates Factors IX and X much more efficiently when associated with a negatively charged phospholipid surface (Ruf, and collaborators, 1991b; Paborsky, et al., 1991).

Despite the significant deterioration of the Clotting capacity of the Truncated Tissue Factor, the Factor of Truncated tissue can promote blood clotting when ata or is functionally associated by some other means with a phospholipid or membrane environment. For example, it is demonstrated in the present description that using a bispecific antibody that binds the Truncated Tissue Factor to a membrane antigen of plasma, restoration of useful coagulant activity is allowed. This led one of the present inventors to develop methods for specific coagulation of vascular tumor in vivo using target constructs to manage Truncated Tissue Factor or variants thereof specifically to tumor vasculation or stroma (WO 96/01653). Intravenous administration of this `` coaguligating '' leads to the location of the coagulants inside of the tumor, thrombosis of the tumor vessels, and resulting necrosis of the tumor

The development of intelligent administration, directed from coagulants to the tumor vasculature, as exemplify using a target antibody composition Bispecific and Truncated Tissue Factor, you can see that It represents an improvement over classical immunotoxin therapy. In fact, this coaguligand treatment induces thrombosis of the tumor vessels in less than 30 minutes, compared to approximately 6 hours needed to achieve the same effect after administration of an immunotoxin. In addition, there is no Notable side effects as a result of the treatment of coaguligating Although the directed administration of such a coagulant as the Truncated Tissue Factor was surprisingly effective, this one still requires the preparation of the `` construction objective''.

Other studies of Tissue Factor with objectives very different have also been released to identify uses of the Truncated Tissue Factor that do not depend on their association with the target substance Regarding this, the Fabric Factor Truncated lately has been considered as a candidate for use in the treatment of disorders such as hemophilia. East work may have developed from attempts to Use tissue apo-factor in these treatments. Apo-TF is a slippery preparation of the Factor of Tissue that was proposed for infusion in hemophiliacs, based on the hypothesis that this spontaneous molecule and preferably it would incorporate or associate with exposed membrane surfaces and available at injury sites. Thus, it was reasoned that the apo-TF could be useful in these treatments, without lead to significant side effects (O'Brien and collaborators, 1988; U.S.A. No. 5,017,556).

Apo-TF therapy has been proposed for use in chronic bleeding disorders characterized by a tendency towards bleeding, both inherited and acquired. U.S.A. No. 5,017,556 describes these disorders as those related to the deficiency of Factors VIII, IX or XI; or those related to the acquisition of inhibitors to Factors V, VIII, IX, XI, XII and XIII. The use of apo-TF, characterized as substantially devoid of the lipid that naturally presents the Tissue Factor and not possessing substantially no procoagulant activity before administration, it is recognized that it is in contrast to the results expected, which has been reasoned to lead to toxicity. Now it seems that the results described in U.S. Pat. number 5,017,556 generally represent an anomaly in the technique, and these studies have been contradicted by others Researchers working in this field.

In fact, during attempts to take the practice studies based on what is described above, it they observed experimental animals to develop effects collaterals such as disseminated intervascular coagulation (DIC). This led to the conclusion that intravenous administration of apo-TF is too dangerous to be used (Sakai and Kisiel, 1990; U.S.A. numbers 5,374,617; 5,504,064; Y 5,504,067).

The development of a soluble, truncated form of Tissue Factor has not been recognized as solving the problems associated with the Tissue Factor or apo-TF. By For example, the Truncated Tissue Factor has been rejected as a alternative to the Fabric Factor, due to the fact that it has been characterized in that it has almost no procoagulant activity when normal plasma test (Paborsky et al., 1991; Patent USES. No. 5,374,617).

Potential uses for the Tissue Factor truncated possible before the present invention were confined then to the targeted administration of the truncated Tissue Factor, for example, using antibodies, and the possible use of the Factor of Truncated tissue to treat a limited number of disorders used in combination with other accessory molecules necessary to the restoration of sufficient activity (U.S. Patent No. 5,374,617). This second possibility has been exploited in certain limited circumstances combining the use of Tissue Factor truncated with coagulation factor administration, Factor VIIa. The combined use of Factor VIIa with the Tissue Factor truncated results in the restoration of sufficient activity coagulant so that this combination is in use to treat blood disorders, such as hemophilia. However, in contrast with the directed administration of coagulants such as the Truncated Tissue Factor discussed in WO 96/01653, the therapy of combination of Truncated Tissue Factor and Factor VIIa not It includes specific direction concepts. This therapy therefore It has been proposed for use only in relation to patients in which coagulation is impaired (U.S. Patent No. 5,374,617; 5,504,064; and 5,504,067).

The most easily identified patient group with these deteriorated coagulation mechanisms are hemophiliacs, including those suffering from hemophilia A and hemophilia B, and those that have high antibody counts directed at the factors of coagulation. In addition, this combined treatment of Tissue Factor truncated and Factor VIIa has been proposed for use in relation to patients suffering from severe trauma, postoperative bleeding or to cirrhosis (U.S. Patent Nos. 5,374,617; 5,504,064; and 5,504,067). Both systemic administration have been proposed by infusion as topical application as useful in these therapies Thus, these therapies can be seen to supplement the body with two `` Factors '' of the coagulation type in order to overcome any natural limitation of these or other related molecules in the coagulation cascade in order to stop bleeding in a specific site

Roy and collaborators have also proposed the use of certain tissue factor mutants in the treatment of myocardial infarction, particularly in the prevention of reocclusion of coronary arteries (U.S. Patent No. 5,346,991). As such, the tissue factor mutants are becoming using as `` thrombolytic substances '', and are described as medicines capable of lysing a thrombus of fibrin-platelet in order to allow the blood flow again through an affected blood vessel. The Tissue Factor mutants described are designated with the intention of being able to neutralize the effects of the Tissue Factor endogenous. Its use in relation to myocardial infarction therapy It is said to allow rapid reperfusion, prevents reocclusion and by therefore limits tissue necrosis.

The artificial elements to recreate the natural environment in the context described above is linked to natural processes, where the Tissue Factor is described as constitutively present in the boundaries between organs in order to allow them to function as a molecule Initiator to stop bleeding. However, this limitation of bleeding episodes in hemophilia naturally need to be achieved without tilting the balance of coagulation paths towards broad coagulation, which would be detrimental to these patients and inhibit the supply of oxygen to the particular tissue or organ in question. Therefore, the wide circulation and activity of the Truncated Tissue Factor would be undesirable and in fact not I would expect it to happen from the studies described previously.

Although the Truncated Tissue Factor has not been previously shown to have no ability to locate preferentially within a given site, and although it is known the coagulant capacity relative to the Tissue Factor is greatly reduced Original, full-size, the present invention demonstrates that when administered systematically to animals with tumors solids, the Truncated Tissue Factor induces specific coagulation of the tumor blood supply, resulting in tumor regression. The different aspects of the present invention therefore they are based on the discovery of selective thrombosis of the tumor vessels by truncated Tissue Factor.

A1. Poor Coagulation Tissue Factor

The surprising finding of the inventors of Truncated Tissue Factor specifically located within the tumor enough to cause antitumor effect, it was discovered during studies using the Truncated Tissue Factor as a control in studies targeting tumors with coagulant antibody (`` coaguligating ''). From this initial discovery, the inventors developed the different aspects of the invention described in the present description. Factor compounds Tissue or constructions for use in the present invention have been developed in this way from the Truncated Tissue Factor Original employee first. In accordance with this, you can now employ several tissue factor constructions, including many Different forms of Truncated Tissue Factor, Tissue Factors longer but still damaged, mutant tissue factors. Any Truncated, Variant or Mutant Modified Tissue Factor or conjugated otherwise to improve your half-life, and all functional equivalents thereof. However, it will be understood that each tissue factor construction for use in the invention is unified by the need to be `` clotting deficient''. As detailed in this description, more ahead, there are several structural considerations that can be employ in the design of the Fabric Factors candidates of poor coagulation, and several tests are available for confirm that the candidate tissue factors are convenient for use in the treatment aspects of this invention. Since technological skills to create a variety of compounds, for example, using molecular biology, and of routine for people with ordinary experience in this technique, and since there is extensive structural and functional guidance released In the present description, the person skilled in the art easily would be able to make and use several Coagulation Tissue Factors deficient different in the context of the present invention.

Also, as described in detail significant in the present description, one or more of a variety of Fabric Factors can also be combined with other substances for use in the advantageous treatment of tumors solids and other diseases associated with fluid vessels prothrombotic In addition to the combination with standard treatments, such as surgery and radiotherapy, the coagulation approach of the The present invention can also be combined with the administration of classic chemotherapeutic drugs other than immunotoxins or coaguligands, or with coagulation factors additional, as exemplified by Factor VIIa.

Since the combined treatments are expected to give an additive, increased or even synergistic antitumor effect, those skilled in the art will also readily appreciate that Tissue Factor constructs that have less than the optimal properties in the types of in vitro assays and in described in the present description can still be used in the context of the present invention. For example, if a candidate deficient Coagulation Tissue Factor construct has coagulant activity towards the lower end of the scale recommended in the present description, such a molecule can still prove that it is useful in combination with coagulation factors, chemotherapies or other substances. anticancer Similarly, constructs of poor coagulation tissue factor candidates that can be considered to have a coagulant activity high enough to cause problems with regard to side effects, can still be shown to be useful after careful live studies using experimental animals and in clinical studies starting with low doses. Therefore, the following guidelines concerning deficient coagulation Tissue Factor molecules are disclosed only as exemplary teachings, and technicians with ordinary experience in the field will readily appreciate that Tissue Factor molecules that do not fit directly within the structural and quantitative guidelines presented in the present description may still have significant therapeutic utility in the context of the present invention. Although determining this fact frequently may generally require live animal testing, these tests are routine for those with ordinary experience in this technique and simply require administration and supervision.

A2. Structural considerations for the factor of poor coagulation growth

Technicians with experience in the technique You will easily appreciate that the Tissue Factor molecules for your use in the present invention cannot be substantially Factor of Original fabric. This is evident since the Tissue Factor original and the nearby variants of it are particularly assets to promote coagulation. Therefore, after the administration to an animal or patient, this would lead to extending the coagulation and would be lethal. Therefore, the Original tissue factor formulations, intact. Likewise not It is believed that attempts to modulate the activity of the Factor of Tissue manipulating your physical environment be particularly productive in the context of the present invention. For example O'Brien's apo-TF approach should be avoided and collaborators (1988) because the DIC is expected to result (`` disseminated intervascular coagulation '').

Figure 2 is disclosed herein. description as an instructional model concerning the domain of the original tissue factor molecule. It is an objective of the invention disclosing Tissue Factor molecules that are not substantially associate with the plasma membrane. Naturally the truncated molecule is the most direct way in which it is achieved a modified Tissue Factor that does not bind to the membrane. These types of truncated constructions are described more completely more ahead. However, the real truncation or shortening of the molecule is not the only mechanism by which you can create Variants of operational tissue factor. As an example only, mutations can be introduced in the C terminal region of the molecule that normally crosses the membrane in order to avoid proper membrane insertion. It is taken into account that the insertion of several additional amino acids, or mutation of these waste already present, can be used to effect such expulsion of membrane. Therefore, modifications with in this regard, they reduce the hydrophobicity of the terminal part C of the molecule so that the thermodynamic properties of this region no longer favorable to membrane insertion.

Considering making structural modifications to the original Tissue Factor molecule, those skilled in the art they will realize the need to maintain significant parts of the molecule enough for the tissue factor variant resulting be able to function to promote at least some coagulation. An important consideration is that the molecule of Tissue Factor should retain substantially its ability to bind to Factor VII or Factor VIIa. Referring to the Figure 2, it will be seen that link region VII / VIIa is generally central to the molecule and this region must therefore be maintained substantially in all variants of Tissue Factor Proposals for use in the present invention. The localization particular of this binding region and the optional use of mutants, either alone or in combination with other substances, it arises in more detail later.

However, certain parts of sequences to from the N-terminal region of the original Fabric Factor also It is considered expendable. Therefore, they can be entered mutations in this region or suppression mutants can be used (truncated from the N-terminal) in the Tissue Factor molecules Candidates for use with this description. Given these guidelines, those skilled in the art will appreciate that the following Truncated, dimeric, multimeric and Tissue Factor constructions mutant by way of example, they are by no means limiting and that many other molecules can be easily prepared and used functionally equivalent.

A3. Coagulation Tissue Factor Constructions deficient as an example.

The following Fabric Factor compositions by way of example, including the truncated, dimeric versions, multimeric and mutated, they can exist as different polypeptides or they can be conjugated to inert carriers, such as immunoglobulins, as described hereinafter description.

i. Truncated Tissue Factor

As used in the present description, the term `` truncated '' when used in relation to the Factor of Fabric means that the construction of a particular Fabric Factor It lacks certain amino acid sequences. The truncated term of This mode means tissue factor constructions of more size short, and differentiates these compounds from other constructions of Tissue Factor that have an association or membrane bond reduced Although the modified tissue factors but substantially full size can thus be consider as functional equivalents of Fabric Factors truncated (`` functionally truncated ''), the term `` truncated '' is used in the present description in its classical sense to mean that the tissue factor molecule becomes deficient in binding with membrane eliminating enough amino acid sequences to make this feature change.

In accordance with the above, a protein of Truncated Tissue Factor or polypeptide is what differs from Factor of original fabric in which a sufficient amount of the sequence of Transmembrane amino acids have been removed from the molecule, in Comparison with the original Fabric Factor. In this context a `` sufficient quantity '' is an amount of amino acid sequence of transmembrane originally sufficient to introduce the Membrane Tissue Factor molecule, or otherwise bond the average functional membrane of the Tissue Factor protein. The elimination of this `` sufficient amount of extension sequence of transmembrane '' therefore creates a protein or a polypeptide of Truncated Tissue Factor deficient in binding capacity with phospholipid membrane, so that the protein substantially It is a soluble protein that does not bind significantly to phospholipid membranes; and that substantially fails to convert the Factor VII in Factor VIIa in a standard Tissue Factor test, and thus retains the so-called catalytic activity that includes Factor X activation in the presence of Factor VIIa. The patent USES. No. 5,504,067 further describes these proteins of Truncated tissue factor.

The preparation of Factor constructions Particular truncated fabrics are described herein. description later. Preferably, the Fabric Factors for use in the present invention they generally lack the transmembrane and cytosolic regions (amino acids 220-263 of SEQ ID NO: 12) of the protein. Without However, there is no need for Tissue Factor molecules Truncated are limited to molecules the size of 219 amino acids. By therefore, constructions between approximately 210 and Approximately 230 amino acids in size can be used. In in particular, the constructions can have approximately 210, 211, 212, 213, 214, 215, 216, 217, 218, 219, 220, 221, 222, 223, 224, 225, 226, 227, 228, 229, or about 230 amino acids of size. Naturally, it will be understood that the intention is to suppress substantially the transmembrane region of approximately 23 amino acids from the truncated molecule. Therefore, in the Truncated Tissue Factor constructions that are longer than from 218 to 222 amino acids in size, the sequence parts significant after that will generally be comprised of approximately 21 amino acids that form the cytosolic domain of the original Tissue Factor molecule. About this, Truncated Tissue Factor constructions may have between approximately 231, 232, 233, 234, 235, 236, 237, 238, 239, 240, or approximately 241 amino acids in size.

In certain preferred embodiments, the Factor Truncated Tissue can be designated as the extracellular domain of the protein of the mature tissue factor. Therefore in Preferred embodiments by way of example, the Tissue Factor truncated can have the amino acid sequence of SEQ ID NO: 1, comprising residues 1-219 of the protein mature (SEQ ID NO: 12). SEQ ID NO: 1 may be encoded by, by example, SEQ ID NO: 10. Of course, SEQ ID NO: 1 is only a Example of Truncated Tissue Factor and any Factor protein Tissue derived from the nucleic acid sequence of SEQ ID NO: 11, or related sequences, which possess the properties desirable high affinity binding with Factor VII or with the Factor VIIa and possess a procoagulation cofactor activity generally reduced will also be useful as described in the present description

ii. Dimeric Tissue Factor Constructions

It has previously been shown that it is possible that the original Tissue Factor on the surface of the cells of the J82 bladder carcinoma exists as a dimer (Fair et al., 1987). The binding of a Factor VII or Factor VIIa molecule to a Tissue Factor molecule can also facilitate the binding of another Factor VII or Factor VIIa to another Tissue Factor (Fair and collaborators, 1987; Bach et al., 1986). In addition, the Factor of Fabric shows structural homology to family members of the cytokine receptor (Edgington et al., 1991) some of which dimerizes to form active receptors (Davies and Wlodawer, 1995). In this way it is taken into account that Truncated Tissue Factor compositions of the present invention They can be useful as dimers.

In accordance with the foregoing, any of the tissue Factor constructs truncated, mutated or otherwise of poor coagulation described in the present description, or a equivalent thereof, can be prepared in a dimeric form for use in the present invention. As is known by the skilled in the art, these Fabric Factor dimers can be prepare using standard techniques of molecular biology and recombinant expression, in which two regions of box coding and are expressed from a vector of expression. Likewise, several technologies of chemical conjugation in relation to the preparation of dimers of Tissue Factor The individual Fabric Factor monomers are they can derive before conjugation. All these techniques would be easily known to those skilled in the art.

If desired, the dimers or multimers of Factor Tissue can be joined by a biologically binding releasable, such as an amino acid link or sequence selectively dissociable. For example, they are taken into account peptide linkers that include a dissociation site for a enzyme preferentially located or active within an environment tumor. Exemplary forms of these peptide linkers are those that are dissociated by urokinase, plasmin, thrombin, Factor IXa, Factor Xa, or a metalloproteinase, such as collagenase, gelatinase or stromelysin.

In certain embodiments, Factor dimers Tissue may further comprise an insertion fraction of hindered hydrophobic membrane, then encourage functional association of the Tissue Factor with the membrane phospholipid, but only under certain defined conditions. How It is described in the context of truncated Tissue Factors, the hydrophobic membrane association sequences are generally stretches of amino acids that promote association with the environment phospholipid due to its hydrophobic nature. Likewise, it they can use fatty acids to provide the insertion fraction of potential membrane. These membrane insertion sequences are they can locate either in term N or in term C of the Tissue Factor molecule, or generally annexed to any another point of the molecule as long as its binding to it does not prevent Functional properties of the tissue factor construction. The attempt of the insertion fraction prevented is to remain no functional until the tissue factor construction is located within the tumor environment, and allow the hydrophobic appendix become accessible and even further promote physical association with the membrane Again, keep in mind that the links biologically releasable and selectively sequences dissociable will be particularly useful in this regard, only the link or sequence being dissociated or otherwise modified after its location inside the tumor environment and exposure to particular enzymes or other molecules bioactive

By way of example only, the inventors have Built Dimeric Truncated Tissue Factor corresponding to a C'-cys-tTF_ {219} dimer (dimer of SEQ ID NO: 3); a dimer of C'-cys-tTF_ {220} (dimer of SEQ ID NO: 6); a dimer of C'-cys-tTF_ {221} (SEQ dimer ID NO: 7); and a number of H_ {6} -N'-cys-tTF_ {219} (dimer of SEQ ID NO: 2). However, it will now be understood that each one of the above sequences are by way of example and of no limiting modes of dimeric structures that can be created and use in accordance with the present invention.

iii. Tri and Tissue Factor Constructions multimerics

In other embodiments, the constructions of Truncated Tissue Factor of the present invention can be Multimeric or polymeric. In this context, a `` construction polymeric '' contains 3 or more Tissue Factor constructions of The present invention. A `` Tissue Factor Construction multimeric or polymeric '' is a construction that comprises a first molecule of tissue factor or operably linked derivative at least a second and a third molecule of Factor of Woven or derived, and preferably, where the construction resulting multimeric or polymeric is still deficient in the coagulant activity compared to the Type Tissue Factor natural. In preferred embodiments, Factor constructions of Mutlimeric and polymeric fabrics for use in this invention they are multimers or polymers of Tissue Factor molecules truncated, which can be optionally combined with others constructs or variants of Coagulation Tissue Factor deficient. The multimers can comprise between about 3 and approximately 20 of these Tissue Factor molecules, being preferably between about 3 and about 15 or about 10 and more preferably between about 3 and about 10. Naturally, the tissue factor multimers of at least about 3, 4, 5, 6, 7, 8, 9 or 10 or so, included within the present invention. Factor units Individual fabrics within multimers or polymers are also they can bind via peptide linkers selectively dissociable or other releasable biological bonds as desired. From new, as with the Fabric Factor dimers presented Previously, constructions can be easily done using either recombinant manipulation and expression or using chemistry standard synthetic.

iv. Factor VII activation mutants

Still other tissue factor constructions useful in the context of the present invention are mutants deficient in the ability to activate Factor VII. the basis of the usefulness of these mutants is in the fact that they are also `` deficient coagulation. '' These `` activation mutants of Factor VII '' is generally defined in the present description as Tissue Factor mutants that link the functional factor VII / VIIa, proteolytically activate Factor X, but are substantially free of ability to activate proteolytically Factor VII. In accordance with the above, these constructions they are tissue factor mutants that lack the activity of Factor VII activation.

The ability of these mutants to activate the Factor VII to function by promoting specific coagulation of tumor, is based both on the location of the construction of the Tissue factor in the vasculature of the tumor, as in the presence of Factor VIIa at low plasma levels. After the administration of this activating mutant Factor VII, the mutant would usually locate inside the vasculature of a tumor vascularized, as any Factor Factor construct would Fabric of the invention. Before localization, the mutant of Tissue Factor would be unable to promote coagulation in any other body site, based on your inability to convert Factor VII to Factor VIIa. However, after the location and accumulation within the region of the tumor, the mutant you will then find enough Factor VIIa of the plasma in order to start the extrinsic coagulation path, leading to the tumor specific thrombosis.

How it develops more completely more hereinafter, the most preferred use of the activation mutants of the Factor VII is in combination with the co-administration of Factor VIIa. Although useful in and out of them, as described above, these mutants will generally have less than a given optimal activity that Factor VIIa is known to be present in plasma only at low levels (approximately 1 nanogram / milliliter, in contrast with approximately 500 nanograms / milliliter of Factor VII in plasma; U.S.A. numbers 5,374,617; 5,504,064; Y 5,504,067). Therefore, the co-administration of Exogenous Factor VIIa together with the Factor activation mutant VII is considerably preferred over the administration of mutants alone. Since these mutants are expected to have almost side effects, their use combined with simultaneous administration, preceding, or subsequent factor VIIa is an aspect particularly advantageous of the present invention.

One or more of a variety of mutants of Factor VII activation can be prepared and used in relation to Some aspect of the present invention. There is a quantity significant scientific knowledge related to tissue factor molecule recognition sites for the Factor VII / VIIa. As an example only, it can be done reference to articles by Ruf and Edgington (1991a), Ruf and collaborators (1992c), and WO 94/07515 and WO 94/28017, each specifically incorporated herein by Reference for additional guidance on these topics. Thus it will be understood that the Factor VII activation region is generally between approximately amino acid 157 and approximately amino acid 167 of the Tissue Factor molecule. However, it is had in note that waste outside this region can also demonstrate that they are important for the activation activity of the Factor VII, and the introduction of mutations in one or more of the waste generally located between approximately amino acid 106 and approximately amino acid 209 of the Fabric Factor sequence (WO 94/07515). In terms from the preferred region, one can generally consider mutating one or more of amino acids 147, 152, 154, 156, 157, 158, 159, 160, 161, 162, 163, 164, 165, 166 and / or 167. With reference to the Preferred candidate mutations generally outside this region, reference can be made to the following substitutions of amino acids: S16, T17, S39, T30, S32, D34, V67, L104, B105, T106, R131, R136, V145, V146, F147, V198, N199, R200 and K201, also considering amino acids A34, E34 and R34 (WO 94/28017).

As indicated, preferably the Tissue factors become deficient in their ability to activate Factor VII by altering one or more amino acids in the region generally between approximately position 157 and approximately position 167 in the amino acid sequence, when done Reference to SEQ ID NO: 12. Mutants by way of example are those where the Trp at position 158 is changed to Arg (SEQ ID NO: 8); where Being in position 162 is changed to Ala; in where Gly at position 164 is changed to Ala (SEQ ID NO: 9); and the double mutant where Trp at position 158 changes to Arg and Ser at position 162 change to Ala. Of course these are mutations. by way of example and any factor mutant is considered of Tissue that has an altered amino acid composition that It has the desirable characteristics of binding to Factor VII / VIIa, but not activating the coagulation cascade, it will be useful in context of the present invention.

A4. In vitro quantitative assessment of the deficiency of coagulant

The tissue factor constructions to use in the present invention, whether truncated, mutated, truncated and mutated, dimeric, multimeric, conjugated with inert carriers to increase your half-life, or any combination of above, each one is deficient coagulation compared to the original natural Fabric Factor. Through the term `` poor coagulation '', as used in the present description, it is understood that the tissue factor constructions have a impaired ability to promote clotting, so that your administration in the systemic circulation of an animal or a human patient does not lead to significant side effects or to limiting toxicity A fabric factor construction can be easily analyze in order to determine if it satisfies this definition, simply conducting a test on an animal experimental. However, the detailed guide is released next to help those skilled in the art in the previous characterization and selection of appropriate candidates from Deficient coagulation tissue factor constructs, with the so that any animal study can be carried out Experimental efficiently and with efficient cost.

In quantitative terms, the Fabric Factors deficient coagulation will be a hundred times or more active than The original, full-size Fabric Factor, that is, will be one hundred times or less capable of inducing plasma coagulation than the original, full-size Fabric Factor, when Test in a suitable phospholipid environment.

More preferably, the Fabric Factors impaired should be a thousand times or less capable of inducing plasma coagulation than the original Type Tissue Factor, of full size, in a suitable phospholipid environment; even more preferably, the Fabric Factors should be ten thousand times or more less capable of inducing plasma coagulation than the Factor of full-size, natural-type fabric in such an environment; Y more preferably, deteriorated Fabric Factors should be one hundred thousand times or less capable of inducing plasma coagulation than its original, full-size Fabric Factor, in an environment suitable phospholipid. It will be appreciated that this expression `` one hundred thousand times '' generally corresponds to one of the constructions currently preferred, the Truncated Tissue Factor of 219 amino acids in size (SEQ ID NO: 1).

Inherent within the definition of `` X times or less able to induce plasma coagulation '' is the concept that the tissue factor under investigation It is still capable of inducing plasma coagulation. Obviously, a Tissue Factor that has been modified to become completely unable to induce coagulation It will generally not be useful in the context of the present invention. Tissue Factors that are less active than the Tissue Factor of the natural type in controlled phospholipid tests by approximately 500,000 times, they are still considered to have utility in relation to the present description. Similarly, all variants and mutants of Tissue Factor that are between about 500,000 times and about a million times less capable of inducing plasma coagulation than its Factor of Original fabric, full size, in a phospholipid environment adequate, they are still considered to have utility in certain realizations However, it is generally considered that a inability of activity one million times (10 6) generally it will be approximately the least active one could Consider for use in the present invention. In addition, those Tissue Factor constructions that are toward the less extreme active in the established range, you can find more utility in relationship with certain defined treatment regimens or in Combined therapies The particular choice of Factor variant Tissue and therapeutic strategy will be easily determined by a skilled in the art.

Regardless of whether there are certain uses preferred and / or optimal and combinations of the different elements of Tissue Factors, Coagulation Tissue Factors deficient for use in the present invention will generally be about 100 times to about a million times less active than the Natural Type Tissue Factor; plus preferably, they will be between about 1000 times and approximately 100,000 times less active; and can be categorized as less active by any number within the ranges established, including approximately ten thousand times. The same ranges can also be varied between approximately 1000 times and a million times, or between 10,000 times and 500,000 times, or something Similary.

One or more of several in vitro plasma coagulation activity assays may be employed in relation to the quantitative test of Failure Coagulation Tissue Factors. For example, a method of conducting an innate plasma coagulation activity assay is as follows:

one)

Add approximately 50 microliters of plasma (human or mouse) to tubes plastic at approximately 37 ° C;

two)

Add approximately 50 microliters of Fabric Factor in size complete re-encoded (preferably from a commercial source, such such as American Diagnostics Inc., Greenwich, CT) to a range of concentrations in a convenient regulator such as free phosphate of calcium or HEPES regulated saline solution, pH 7.4 at 37 ° C. Add to other tubes the candidate of Tissue Factor in truncated version or mutant at a range of concentrations in it regulator.

3)

Add approximately 50 microliters of 30 mM CaCl 2 at about 37 ° C;

4)

record time for the first strands of fibrin to form; Y

5)

 build a Standard concentration curve of Fabric Factor size complete (mol per liter) with respect to coagulation time. Construct a concentration curve of Mutant Tissue Factor candidate (mol per liter) with respect to coagulation time. Calculate the difference in activity between the Tissue Factor of Full size and the `` Test '' Tissue Factor comparing the necessary concentration of each to give a time of coagulation equivalent to about half of the maximum decrease in coagulation time. The mutant tissue factor `` test '' must be more than 100 times less capable than the Factor of full-size tissue on a molar base to induce plasma coagulation

Variations of this type can be carried out. trial, as would be evident to an experienced person ordinary in the art. For example, they can be carried out tests based on the union of the Tissue Factor and the construction of Candidate Tissue Factor to a cell membrane or surface phospholipid, for example, using an antibody or other ligand to make this union. In these tests, the candidate or Factor of Truncated test tissue or tissue factor mutant should be more than 100 times less effective in inducing coagulation of the plasma that the tissue factor of natural type, when joined by medium of an antibody or other ligand to a cell membrane or phospholipid surface. With the tissue factor mutants that they do not allow Factor VII to efficiently become Factor VIIa, it may be necessary to add Factor VIIa to the plasma to Get this level of activity. In an example essay, this Activity can be measured using the following method:

one)

Cells are incubated such as A20 mouse lymphoma cells (I-A d positive) (for example 4 x 10 6 cells / milliliter, 50 microliters) in a regulator, such as phosphate regulated saline solution, for approximately one hour at about room temperature with a substance binding promoter, such as a bispecific antibody (50 micrograms / milliliter, 25 microliters), for example, in terms of A20 cells, which consist of a Fab 'arm of such an antibody as the B21-2 antibody directed against I-A d, linked to the Fab 'arm of a antibody such as the 10H10 antibody directed against an epitope non-inhibitory in Tissue Factor;

two)

Prepare a identical set of tubes containing cells, but no bispecific antibody or other binding substance (`` tethering '');

3)

Wash the cells effectively, for example, twice at room temperature, and resuspend the cells in approximately 50 microliters of phosphate regulated saline solution;

4)

Add variant concentrations of the tissue factor mutants Candidates in phosphate regulated saline (approximately 50 microliters) at approximately room temperature. The bispecific antibody or other binding substance captures the mutant of the Tissue Factor and puts it in close approximation with the cell surface Factor VIIa (1-10 is added nM) in addition to the tissue factor mutant when desired for determine activity in the presence of Factor VIIa. It fits the total volume per tube at approximately 150 microliters with phosphate regulated saline solution. The tubes are incubated during about an hour at about temperature environment;

5)

They heat up cells at approximately 37 ° C.

6)

Chloride is added calcium (approximately 50 mM, 50 microliters) and mouse plasma or human citrate (approximately 50 microliters) to approximately 37 ° C

7)

The time for the first strands of fibrin to form; Y

8)

The chart is drawn of the coagulation time (in seconds) with respect to the Tissue Factor mutant concentration (mol per liter) for Coated or uncoated cells with binding substance, by example, bispecific antibody. The concentration of Tissue Factor mutant that gives a clotting time equivalent to about half of the maximum decrease in the clotting time (usually 50 to 100 seconds). I know calculates the increase in coagulation activity given by the bispecific antibody and should be 100 times more.

Factor Factor compositions are considered Tissue candidates prepared by the present invention can be test using tests similar to those described above to confirm that its functionality has been maintained, but that its ability to promote coagulation has deteriorated in how minimum the required amount of approximately 100 times and preferably about 1,000 times, more preferably in about 10,000 times, and more preferably in Approximately 100,000 times

In embodiments where it is taken into account that an additional substance should finally be used in combination with the candidate deficient coagulation Tissue Factor, it is important that the additional factor or substance be included in the in vitro assay. A particularly relevant example is the analysis of a Factor VII activation mutant, which should preferably be analyzed together with the addition of Factor VIIa. However, Factor VIIa is not the only additional component that can be tested in this way. In general, additional substances can be called `` additional candidates. '' To identify an additional candidate, or to optimize preferred amounts of the candidates for use in the present invention, tests such as those described above in parallel can be carried out. That is, coagulation would be measured or determined in the absence of the additional candidate, and then the candidate substance would be added to the composition and the time and / or duration of blood coagulation would be redetermined. An additional candidate substance that works in combination with a Tissue Factor mutant or variant would result in an overall level of coagulation between about 100 times and about 1,000,000 times lower than that observed with the original Tissue Factor, again it would be a combination suitable for use in the context of the present invention.

Technicians with ordinary experience in the field will understand that each of the previous in vitro tests and variations thereof are relatively simple to establish and perform. In this way, a panel of candidate Tissue Factor variants and combinations of Tissue Factors with other substances can be tested and the most promising candidates can be selected for further studies, particularly for experimental tests in an animal or human trial.

Regardless of whether it is believed that the above tests are particularly useful in relation to the present invention, in vitro tests intended for use with the present disclosure are not limited to these tests. In accordance with the foregoing, any desired coagulation or procoagulation assay can be carried out. For example, for more details regarding the Truncated Tissue Factor and procoagulation tests, experts refer to US Pat. Nos. 5,437,864; 5,223,427; and 5,110,730 and the International Patent with publication numbers WO 94/28017; WO 94/05328; and WO 94/07515, each of which completes the present description with respect to the tests.

TO 5. In vivo confirmatory studies

Those skilled in the art will understand that Coagulation Factor Factor deficient mutants candidates, variants or combinations thereof with additional substances, generally they must be tested on a live stage before be used on a human subject These preclinical tests in animals They are routine in the art. To carry out these tests Confirmatory, all that is required is an animal model accepted in the technique of the disease in question, such as a animal that has a solid tumor. Any animal can be used in this context, such as, for example, a mouse, rat, rabbit Indian, hamster, rabbit, dog, chimpanzee, or similar. In the context of cancer treatment, studies using animals small, such as mice, are widely accepted because they are predictive of clinical efficacy in humans, and these models of animals are therefore preferred in the context of the present invention, since they are readily available and relatively little expensive, at least compared to other animals Experimental

The way to carry out an animal test experimental will be directly that of ordinary experience in the technique. All that is required to carry out this test is establish equivalent treatment groups, and administer the test compounds to a group while several parallel control studies in equivalent animals in the remaining group or groups. Animals are monitored during course of study and finally animals are sacrificed for Analyze the effects of treatment.

One of the most useful features of the Present invention is its application to the treatment of tumors vascularized In accordance with the above, they can be carried conduct antitumor studies to determine specific thrombosis within the vasculature of the tumor and the overall antitumor effects. As part of these studies, you can also monitor the specificity of effects, including evidence of coagulation in other vessels and tissues and the good general condition of the animals is You must monitor carefully.

In the context of the treatment of tumors solid, it is taken into account that the constructions of Factor of Effective fabric and effective construction quantities will be those constructions and quantities that result generally at least about 10% of the vessels inside of a vascularized tumor that exhibits thrombosis, in the absence of significant thrombosis in vessels outside the tumor; preferably the thrombosis will be observed at least about 20%, approximately 30%, approximately 40%, or approximately 50% also of blood vessels within the tumor mass solid, without significant localized thrombosis. In the treatment of large tumors, the present inventors have observed Routinely these positive effects. Without a doubt, they have been analyzed tumors in which at least about 60%, about 70%, about 80%, about 85%, approximately 90%, approximately 95% or up to and including approximately 99% of the tumor vessels have become thrombotic Naturally, the greater the number of vessels it exhibits thrombosis, treatment is more preferred, as long as the effect continues being specific, relatively specific or preferential to the vasculature associated with the tumor and as long as the coagulation is not apparent in other tissues to a degree sufficient to cause damage significant to the animal.

After induction of thrombosis within the blood vessels of the tumor, the tissues of the tumor become necrotic The successful use of the constructions to be used in the invention, or the doses thereof, can also be assessed in terms of the expansion of specifically induced necrosis in the tumor Again, the expansion of cell death in the tumor will be assessed in relation to the maintenance of healthy tissues in All other areas of the body. Factor substances Optimal tissue, combinations or doses will have therapeutic utility according to the present invention when its administration gives as a result at least about 10% of the tissue in the tumor becomes necrotic (10% of necrosis). Again, it is preferable. obtain at least about 20%, about 30%, approximately 40% or approximately 50% necrosis in the tumor region, without significant adverse side effects. These beneficial effects have been observed again by present inventors. Naturally, it will be preferable to use constructions and doses capable of inducing at least 60%, approximately 70%, approximately 80%, approximately 85%, approximately 90%, approximately 95%, up to including 99% of tumor necrosis, while constructions and doses used do not result in effects significant collaterals or some other unwanted reactions in the animal.

All the above determinations can be easily do and properly value by technicians with ordinary experience in the field. For example, scientists and attending physicians could use this data from experimental animals in dose optimization suitable for The human treatment In subjects with advanced disease, it can be tolerate some degree of side effects. However, the patients in the early stages of the disease can be treated with more moderate doses in order to obtain a therapeutic effect significant in the absence of side effects. The effects observed in these studies of experimental animals should be preferably statistically significant on the levels of control and should be reproducible from study to study.

People with ordinary experience in the technique will also understand that Factor Factor constructions Tissue, combinations and doses that result in thrombosis tumor-specific and necrosis towards the lower end of the effective ranges mentioned above, still however they may be useful in relation to the present invention. By example, in embodiments where an application is taken into account continuous active substances, an initial dose of a construction that only results in approximately 10% of thrombosis and / or necrosis will however be useful, particularly as It is often observed that this initial reduction `` primes '' the tumor for a subsequent destructive assault after the subsequent reapplication of therapy. In any case, even if exceeds approximately 40% or so tumor inhibition is not finally achieves (which is the general goal), it will be understood that any induction of thrombosis and necrosis is useful because represents an advance on the patient's condition before treatment.

As discussed above in relation to the in vitro test system, it will naturally be understood that combinations of substances intended for use together should be tested and used together. By way of example only, the Factor VIIa activation mutant of the present invention is included in this category and should generally be tested together with the simultaneous, previous or subsequent administration of exogenous Factor VIIa. Similarly, the individual Tissue Factor constructs of the present invention can be analyzed directly in combination with one or more chemotherapeutic drugs, immunotoxins, coaguligands or the like. Analyzes of the combined effects of these substances would be determined and assessed according to the guidelines presented above.

A6 Biologically Functional Equivalents

As discussed, Factor's compositions Truncated fabric useful in the present invention are those that they will generally promote coagulation at least 100 times less effectively that the natural type of Tissue Factor. In others embodiments, the Truncated Tissue Factor promotes coagulation at least 1,000 times less effectively, in other embodiments Truncated Tissue Factor promotes coagulation as a minimum 10 4 or up to 10 5 times less effectively than the Factor of Fabric of the natural type, being the Fabric Factors that are of approximately 10 6 times or less active than the Factor of Natural type fabric approximately the minimum activity intended required.

The Fabric Factors by way of example are those lacking transmembrane and cytosolic region (amino acids 220-263). A Truncated Tissue Factor of the present invention is given in SEQ ID NO: 1 and contains the amino acids 1-219 of the Natural Type Tissue Factor (SEQ ID NO: 12). Of course this is only an example of Factor of truncated fabric and other constructions of Truncated Fabric Factor, for example, a construction that comprises amino acids 1-220; 2-219, 3-219 or any other truncated from identification sequence number 12 that leaves the molecule lacking the transmembrane domain and / or domains Cytosolic Tissue Factor of the natural type otherwise resulting in a functionally comparative molecule. Mutants are also taken into account, as described in detail previously.

Using the detailed guide released previously, still other equivalents of the Fabric Factors were they can do. Modifications and changes can be made to the structure of the Tissue Factor and still get a molecule that have similar or otherwise desirable characteristics. By For example, certain amino acids can be substituted for others amino acids in a protein structure without appreciable loss of interactive linking capability, such as linking to Factor VIIa. Since this is the interactive and natural capacity of a protein that defines that biological functional activity of the protein, certain sequence substitutions of amino acids in a protein sequence (or of course, the underlying DNA sequence) and yet get a protein with equal properties (agonistic). This way you have in account that several changes can be made in the Factor sequence of Tissue (SEQ ID NO: 12) proteins and peptides (or DNA sequence underlying, SEQ ID NO: 11) without appreciable loss of utility or biological activity

The experienced technician also understands that, inherent in the definition of a biologically protein or peptide functional equivalent, there is the concept that there is a limit to number of changes that can be made within a defined part of the molecule and still get as a result a molecule with a Acceptable level of equivalent biological activity. Peptides Biologically functional equivalents are thus defined in the present description as the peptides in which certain Amino acids, not most or all, can be substituted. Since then, you can easily make a plurality of different proteins / peptides with different substitutions and use them according with the invention

Amino acid substitutions are usually based on the relative similarity of chain substituents amino acid side, for example, its hydrophobicity, hydrophilicity, load, size, and the like. An analysis of the size, form and type of amino acid side chain substituents reveals that arginine, lysine and histidine are charged residues positively; that alanine, glycine and serine are one size Similary; and that phenylalanine, tryptophan and tyrosine have Generally similar form. Therefore, based on these considerations, arginine, lysine and histidine; alanine, glycine and serine; and phenylalanine, tryptophan and tyrosine; are defined in the present description as biologically equivalent functional.

By making more quantitative changes, you can Consider the hydropathic index of amino acids. To each amino acid has been assigned a hydropathic index based on its hydrophobicity and load characteristics, these are: isoleucine (+4.5); valine (+4.2); leucine (+3.8); phenylalanine (+2.8); cysteine / cystine (+2.5); methionine (+1.9); Alanine (+1.8); glycine (-0.4); threonine (-0.7); serine (-0.8); tryptophan (-0.9); tyrosine (-1.3); proline (-1.6); histidine (-3.2); glutamate (-3.5); glutamine (-3.5); aspartate (-3.5); asparagine (-3.5); lysine (-3.9); and arginine (-4.5).

The importance of the amino acid index hydropathic to confer interactive biological function on a Protein is generally understood in the art (Kyte and Doolittle, 1982, incorporated herein by reference). It is known that certain amino acids can be substituted for others amino acids that have a similar hydropathic index or rating and still retain a similar biological activity. When making changes based on the hydropathic index, substitution of amino acids whose hydropathic indices are within ± 2, those within ± 1 are particularly preferred, and those within ± 0.5 are still more preferred particularly.

It is understood that an amino acid can be substituted on the other that has a similar hydrophilicity value and continues obtaining a biologically equivalent protein. As detailed in U.S.A. No. 4,554,101 (incorporated herein description by reference), the following have been assigned hydrophilicity values to amino acid residues: arginine (+3.0); lysine (+3.0); aspartate (+3.0 ± 1); glutamate (+3.0 ± 1); serine (+0.3); asparagine (+0.2); glutamine (+0.2); glycine (0); threonine (-0.4); proline (-0.5 ± 1); to the girl (-0.5); histidine (-0.5); cysteine (-1.0); methionine (-1.3); valine (-1.5); leucine (-1.8); isoleucine (-1.8); tyrosine (-2.3); phenylalanine (-2.5); Tryptophan (-3.4).

When making changes based on the values of hydrophilicity, the substitution of amino acids whose hydrophilicity values are within ± 2, those that are within ± 1 are particularly preferred, and those that are within ± 0.5 they are still more particularly preferred.

B. Tissue Factor Polynucleotides B1. DNA segment

The polynucleotides, which encode the Factors Tissue of the present invention can encode a protein of Whole tissue factor, as long as it is deficient coagulation, a Protein domain of Functional Tissue Factor, or any mutant or variant tissue factor polypeptide according to the Detailed guide presented in this description. If one wishes prepare a Truncated Tissue Factor, a mutant Tissue Factor or a Truncated and mutated Tissue Factor, the useful DNA segment underlying and the gene will generally be the same. Since the DNA Human is available for the whole Tissue Factor molecule, it will generally be preferred to use this human construction, since It aims to clinical treatment in humans. However, of none The use of other Tissue Factor genes is excluded, in both the protein produced does not produce immune reactions or adverse effects after administration to a human patient. The methods and compositions described in U.S. Pat. number 5,110,730 are specifically incorporated in this description by reference for the purposes of complementing additionally the description of the applicants with reference to the genes and segments of DNA for use with it.

The polynucleotides can be derived from DNA genomic, that is, cloned directly from the genome of a particular organism In other embodiments, however, the Polynucleotides can be complementary DNA (cDNA). The cDNA is DNA prepared using messenger RNA (mRNA) as a standard. In this way, a cDNA does not contain any interrupted coding sequence and it usually contains almost exclusively the region or regions of coding for the corresponding protein. In others embodiments, the polynucleotide can be synthetically produced. As is known to those skilled in the art, it is generally prefers to use the cDNA construct in recombinant expression, since these constructions are easy to handle and use. The use of longer genomic clones, up to and including sequences of Full size, in no way is excluded.

Although an amazing feature of the present invention is that the tissue factor constructs preferentially they are located specifically in the vasculature of a solid tumor and induce specific antitumor effects in it, It is also taken into account that the tissue factor proteins and polypeptides can be administered to the tumor environment using a recombinant vector that expresses the products of the Factor of Tissue. These `` gene therapy '' approaches to treatment against cancer can be easily practiced by referring to certain scientific references that refer to constructions and appropriate protocols. By way of example only, it can be used a viral vector, such as a retroviral vector, herpes virus simplex, HSV (U.S. Patent No. 5,288,641), cytomegalovirus; adeno-associated virus, VAA (U.S. Patent No. 5,139,941); and / or a adenoviral vector.

The genomic human DNA sequence for the Tissue Factor is disclosed in SEQ ID NO: 11, with the corresponding amino acid sequence that is disclosed in the SEQ ID NO: 12. If one wishes to express Factor VII, the DNA and the amino acid sequences are disclosed in SEQ ID NO: 13 and SEQ ID NO: 14 respectively.

It is taken into account that natural variants of the Tissue Factor exist and have different sequences that those described in this description. Thus, the present invention is not limited to the use of the polynucleotide sequence provided for the Tissue Factor but, instead, includes the use of any variant that occurs naturally. The The present invention also encompasses chemically synthesized mutants. of these sequences, intelligently designed after a application of structural and functional considerations quantified detailed above.

Another class of sequence variants are result of the codon variation. Because there are several codons for most of the normal 20 amino acids, many DNA Different may encode the Tissue Factor. The reference to the Table I will allow these variants to be identified.

TABLE I

       \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {lllllllll} \ hline
 Amino acids \ + \ multicolumn {8} {c} {Codons} \\\ hline Alanina \ qquad
\ + \ qquad Ala \ + A \ + GCA \ + GCC \ + GCG \ + GCU \ + \ + \\
Cysteine \ qquad \ + \ qquad Cys \ + C \ + UGC \ + UGU \ + \ + \ + \ + \\
Aspartic Acid \ qquad \ + \ qquad Asp \ + D \ + GAC \ + GAU \ + \ + \ +
\ + \\ Glutamic Acid \ qquad \ + \ qquad Glu \ + E \ + GAA \ + GAG
\ + \ + \ + \ + \\ Phenylalanine \ qquad \ + \ qquad Phe \ + F \ + UUC \ +
UUU \ + \ + \ + \ + \\ Glycine \ qquad \ + \ qquad Gly \ + G \ + GGA \ +
GGC \ + GGG \ + GGU \ + \ + \\ Histidine \ qquad \ + \ qquad His \ + H
\ + CAC \ + CAU \ + \ + \ + \ +
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
    

TABLE I (continuation)

       \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {lllllllll} \ hline
 Amino acids \ + \ multicolumn {8} {c} {Codons} \\\ hline Isoleucine
\ qquad \ + \ qquad Ile \ + I \ + AUA \ + AUC \ + AUU \ + \ + \ + \\
Lysine \ qquad \ + \ qquad Lys \ + K \ + AAA \ + AAG \ + \ + \ + \ + \\
Leucine \ qquad \ + \ qquad Leu \ + L \ + UUA \ + UUG \ + CUA \ + CUC
 \ + CUG \ + CUU \\ Methionine \ qquad \ + \ qquad Met \ + M \ + AUG \ +
\ + \ + \ + \ + \\ Asparagine \ qquad \ + \ qquad Asn \ + N \ + AAC \ +
AAU \ + \ + \ + \ + \\ Prolina \ qquad \ + \ qquad Pro \ + P \ + CCA \ +
CCC \ + CCG \ + CCU \ + \ + \\ Glutamine \ qquad \ + \ qquad Gln \ + Q
\ + CAA \ + CAG \ + \ + \ + \ + \\ Arginine \ qquad \ + \ qquad Arg \ + R
\ + AGA \ + AGG \ + CGA \ + CGC \ + CGG \ + CGU \\ Serina \ qquad \ +
\ qquad Ser \ + S \ + AGC \ + AGU \ + UCA \ + UCC \ + UCG \ + UCU \\
Threonine \ qquad \ + \ qquad Thr \ + T \ + ACA \ + ACC \ + ACG \ + ACU
\ + \ + \\ Valine \ qquad \ + \ qquad Val \ + V \ + GUA \ + GUC \ + GUG
 \ + GUU \ + \ + \\ Tryptophan \ qquad \ + \ qquad Trp \ + W \ + UGG \ +
\ + \ + \ + \ + \\ Tyrosine \ qquad \ + \ qquad Tyr \ + Y \ + UAC \ + UAU
\ + \ + \ + \ +
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
    

B2. Mutagenesis

Specific site mutagenesis is a technique useful in the preparation of individual peptides, or proteins or biologically functional equivalent peptides, through the specific mutagenesis of the underlying DNA. The technique also gives know an easy ability to prepare and test variants of sequence, which incorporates one or more of the considerations above, introducing one or more sequence changes of DNA nucleotides. Specific site mutagenesis allows mutant production through the use of sequences from specific oligonucleotides encoding the DNA sequence of the desired mutation, as well as a sufficient number of nucleotides adjacent, to provide a size-priming sequence sufficient and sequence complexity to form a stable duplex on both sides of the suppression joint that is going through. Typically, a primer of about 17 to 25 is preferred nucleotides in size, with approximately 5 to 10 residues in both sides of the union of the sequence being altered.

The technique of site specific mutagenesis It is well known in the art. As will be appreciated, the technique typically employs a bacteriophage vector that exists in both form single chain as double chain. Typical useful vectors in site-directed mutagenesis include vectors such as the phage M13. These phage vectors are commercially available and their Usage is generally well known to those skilled in the art. Double chain plasmids are also routinely used. in site-directed mutagenesis, which eliminates the stage of transfer the gene of interest from a phage to the plasmid.

In general, site-directed mutagenesis is performed by first obtaining a single chain vector, or by fusing two double chain vector chains that include within their sequence a DNA sequence encoding the desired protein. An oligonucleotide primer having the desired mutated sequence is prepared synthetically. This primer is then re-associated with the preparation of single-stranded DNA, and is subjected to DNA polymerization enzymes such as E. coli Klenow polymerase fragment, in order to complete the synthesis of the chain that carries the mutation. . Thus, a heteroduplex is formed in which one chain encodes the original non-mutated sequence and the second chain carries the desired mutation. This heteroduplex vector is then used to transform suitable cells, such as E.coli cells,} and clones that include recombinant vectors that carry the mutated sequence configuration are selected.

The preparation of gene sequence variants selected using site directed mutagenesis is disclosed as a means of producing potentially useful species and not supposed to be limiting, since there are other ways in which they can obtain sequence variants of the genes. For example, the Recombinant vectors encoding the desired gene can be treated. with mutagenic substances, such as hydroxylamine, to obtain sequence variants. Convenient techniques are also described. in U.S.A. No. 4,888,286, incorporated herein Description by reference.

Although the above methods are convenient for use in mutagenesis, the use of the chain reaction of Polymerase (PCR MR) is generally preferred now. Is technology offers a fast and efficient method to introduce the desired mutations in a given DNA sequence. The following text particularly describes the use of the chain reaction of polymerase to introduce point mutations in a sequence, since which can be used to change the amino acid encoded by the given sequence The adaptations of this method are also suitable for introducing restriction enzyme sites in a DNA molecule

In this method, oligonucleotides are designed synthetic to incorporate a point mutation at one end of a amplified segment After the chain reaction of polymerase, the amplified fragments are blunted by treating them with fragments of Klenow, and the blunt fragments are linked and subclone into a vector to facilitate sequence analysis.

To prepare the desired standard DNA mutagenize, the DNA is subcloned into a copy number vector high, such as pUC19, using flanking restriction sites the area that is going to mutate. The standard DNA is prepared using a plasmid minipreparation. Oligonucleotide primers suitable that are based on the parent sequence, but that contain the desired point mutation and that are flanked at the 5 'end through a restriction enzyme site, they are synthesized using a automatic synthesizer It is generally required that the primer be homologous to the standard DNA for approximately 15 bases or so. The primers can be purified by gel electrophoresis of denaturing polyacrylamide, although this is not absolutely necessary for use in the polymerase chain reaction. The 5 'end of the oligonucleotides should then be phosphorylated

The standard DNA should be amplified by polymerase chain reaction, using primers oligonucleotides containing the desired point mutations. The MgCl2 concentration in the regulated amplification solution It will generally be about 15 mM. Generally they should 20 to 25 chain reaction cycles of polymerase as follows: denaturation, 35 seconds at 95; hybridization, 2 minutes at 50 ° C; and extension, 2 minutes at 72 ° C. The polymerase chain reaction will usually include a last extension cycle of approximately 10 minutes at 72 ° C. After the final stage of extension, approximately 5 units of Klenow fragments should be added to the reaction mixture and incubate for another 15 minutes at approximately 30 ° C. The exonuclease activity of Klenow fragments is required to make the ends wash and are convenient for cloning blunted.

The resulting reaction mixture generally must be analyzed by agarose gel electrophoresis or non-denaturing acrylamide to verify that amplification has produced the predicted product. Then we would proceed to the reaction mixture eliminating most oils minerals, extracting with chloroform to remove oil remaining, extracting with regulated phenol and then concentrating by precipitation with 100% ethanol. Then they should digest about half of the amplified fragments with a restriction enzyme that cuts all sequences of flank used in oligonucleotides. The digested fragments are Purify on a low gelation / fusion agarose gel.

To subclone the fragments and verify the point mutation, the two amplified fragments would be subcloned into a suitable digested vector by blunt ligation. This would be used to transform E. coli , from which the plasmid DNA could subsequently be prepared using a mini preparation. The amplified part of the plasmid DNA would be analyzed by DNA sequencing to confirm that the correct point mutation was generated. This is important since Taq DNA polymerase can introduce additional mutations in DNA fragments.

The introduction of a point mutation also can be performed using polymerase chain reaction steps sequential In this procedure, the two fragments that encompass the mutation is fixed to each other and spread by synthesis mutually initiated This fragment is then amplified by a second stage of polymerase chain reaction, avoiding by this the blunt ligation required in the protocol previous. In this method, the preparation of the standard DNA, the generation of oligonucleotide primers and the first Polymerase chain reaction amplification are performed as It has been described above. However, in this process the Oligonucleotides chosen should be homologous to the standard DNA over a stretch of approximately 15-20 bases and must also overlap with each other for approximately ten bases or more.

In the second chain reaction amplification of polymerase, each amplified fragment and each primer would be used of flank sequence and chain reaction of polymerase for between about 20 and about 25 cycles, using the conditions described above. From again the fragments would be subcloned and it would be verified that the punctual mutation was correct by using the stages noted above.

Using any of the above methods, it is generally preferred to introduce the mutation by amplifying a fragment as small as possible. Of course, parameters such as the fusion temperature of the oligonucleotide, will generally be influenced by the GC content and the size of the oligo, should also be carefully considered. The execution of these methods, and their optimization if necessary, will be known to those skilled in the art, and is further described in several publications, such as Current Protocols in Molecular Biology, 1995 .

B3 Expression and production constructions of protein

Throughout this application, the term `` construction of expression '' means that it includes any type of construction genetics that contains a nucleic acid that encodes a product of gene in which part or all of the sequence encoding the acid Nucleic is capable of being transcribed. The transcription usually It will be translated into a protein. In this way, the expression preferably includes both the transcription of a Factor gene of Tissue as the translation of a factor of a Factor mRNA Woven into a tissue factor protein product.

A technique frequently employed by experts in the field of protein production today is get the so-called `` recombinant '' version of the protein, to express it in a recombinant cell and get the protein from of these cells. These techniques are based on the `` cloning '' of a DNA molecule that encodes the protein from a library of DNA, that is, obtaining a different specific DNA molecule from other parts of the DNA. This can be achieved by, for example, clone a cDNA molecule, or clone a DNA molecule similar to genomic Techniques such as these would be suitable for production of particular Fabric Factor compositions of according to the present invention. Fusion proteins recombinants are discussed in greater detail later in the present description, and U.S. Pat. Number 5,298,599 is example in addition to the production and use of fusion protein.

For the expression of the Tissue Factor, as a suitable clone or clones (full size if desired), whether based on cDNA or genomic, can be continued preparing an expression system for recombinant preparation of the Fabric Factors. The engineering of DNA segments for expression in a prokaryotic or eukaryotic system you can carry out by means of techniques generally known for experts in the field of recombinant expression. It is believed that virtually any expression system can be used in the Expression of these proteins.

These proteins can be successfully expressed in eukaryotic expression systems, for example, CHO cells, as described by Rehemtulla et al. (1991), however, it is considered that bacterial expression systems, such as pQE-60 of E Coli will be particularly useful for large-scale preparation and subsequent purification of proteins or peptides. The cDNAs for the Tissue Factor can be expressed in bacterial systems, the encoded proteins being expressed as fusions with β-galactosidase, ubiquitin, Schistosoma japonicum glutathione S-transferase and the like. It is believed that bacterial expression will have advantages over eukaryotic expression in terms of ease of use and quantity of materials obtained therefrom. The techniques of US Patent Nos. 5,298,599 and 5,346,991 are also incorporated herein by reference to further complement the production methods of the soluble Tissue Factor described in the present description, particularly incorporating US Patent No. 5,346,991 for the purpose of further complementing the description with respect to the creation and production of mutants and variants of the Tissue Factor.

In order for the construction to carry out the expression of a tissue factor transcript, the polynucleotide encoding the Fabric Factor polynucleotide will be under the transcription control of a promoter. A `` promoter '' refers to a DNA sequence recognized by the synthetic machinery of the host cell, or synthetic machinery entered, which is required to start transcription specific to a gene. The phrase `` under transcription control '' means that the promoter is in the right place in relation to the polynucleotide to control the initiation and expression of polynucleotide RNA polymerase. The term promoter will be used here to refer to a group of control modules of transcript that are grouped around the initiation site of RNA polymerase II.

In terms of microbial expression, the U.S.A. 5,583,013; 5,221,619; 4,785,420; 4,704,362; and 4,366,246 further complement the present description in relationship with gene expression in host cells recombinant

B4 Tissue Factor Purification and compositions related

As soon as the peptides have been expressed, they can isolate and purify using purification techniques of proteins well known to those skilled in the art. These compositions will be used alone or in combination with antibodies, chemotherapies and effector ligands as therapeutic substances in Tumor treatment as detailed later in the present description The peptides by way of example of the The present invention is shown in SEQ ID NO: 1 - SEQ ID NO: 9, of course it is understood that these are only examples and also takes into account any mutation, alteration or variants that are naturally present these sequences, as useful in conjunction with the present invention.

Protein purification techniques are very known to experts in the field. These techniques tend to involve the fractionation of the cellular medium to separate the protein of interest of other components of the mixture. Methods analytical particularly suitable for the preparation of a pure peptide are ion exchange chromatography, exclusion chromatography, polyacrylamide gel electrophoresis, isoelectric approach and the like. A particularly method efficient to purify peptides is the liquid chromatography of fast protein or even high-performance liquid chromatography Pressure.

Several other techniques suitable for use in Protein purification will be well known by experts in field. These include, for example, precipitation with sulfate of ammonium, PEG, antibodies and the like or by denaturation by heat, followed by centrifugation; such chromatography stages as ion exchange, gel filtration, reverse phase, hydroxylapatite and affinity chromatography; isoelectric focus; gel electrophoresis; and combinations of these and other techniques. As is generally known in the art, it is believed that it can be change the order of conducting the different stages of purification, or that certain stages can be omitted, and anyway it would have as result a convenient method for the preparation of a protein or substantially purified peptide.

As described in the present description in detail, generally preferred techniques to purify Tissue Factor constructs expressed for use in the present invention comprise the generation of a molecule of Tissue Factor that includes a purification label of affinity and the use of an affinity separation matrix for get the construction of free tissue factor from the majority or from All polluting species. Many of these tags from fusion proteins are known to experienced technicians ordinary in the field and these expression and separation protocols They can be carried out easily. There is also technology available to dissociate the original affinity tag before use of the released protein or polypeptide, which can be done inserting a sensitive protease linker between the label of affinity and protein of interest. This methodology will certainly be used in relation to aspects of the present invention. The patent USES. No. 5,298,599 is also instructive regarding this. Without However, it is also known that many of these labels do not prevent ability of the expressed protein to carry out its functions biological, and label removal is not required necessarily before the use of the tissue factor construction in the present invention.

C. Pharmaceutical compositions and equipment

The pharmaceutical compositions herein invention will generally comprise an effective amount of Factor of truncated tissue dissolved or dispersed in a carrier Pharmaceutically acceptable or aqueous medium.

The phrases `` pharmaceutically or pharmacologically acceptable '' refer to molecular entities and compositions that do not produce an adverse reaction, allergic or otherwise contrary when administered to an animal, or a human, as Appropriate. As used in this description, `` carrier pharmaceutically acceptable '' includes any and all solvents, dispersion media, coatings, substances antibacterial and antifungal, isotonic substances and retardants of Absorption and the like. The use of these means and substances for Active pharmaceutical substances is well known in the art. Except to the extent that any conventional means or substance is incompatible with the active ingredient, its use is considered in therapeutic compositions Can also be incorporated supplementary active ingredients in the compositions.

C1. Parenteral formulations

The Truncated Tissue Factor of this invention will frequently be formulated for administration parenteral, for example, formulated for intravenous injection, intramuscular, subcutaneous or other of these routes, including direct instillation in a tumor or diseased site. The preparation of an aqueous composition containing a coagulant substance targeting the tumor as an active ingredient will be known by the skilled in the art in light of the present description. Typically, these compositions can be prepared as injectables, either as solutions or liquid suspensions; too solid forms suitable for use for prepare solutions or suspensions after the addition of a liquid before injection; and preparations can also be emulsify

The solutions of active compounds such as free base or pharmacologically acceptable salts can be prepared in water conveniently mixed with a surfactant, such as hydroxypropyl cellulose. Glycerol dispersions can be prepared, liquid polyethylene glycols, and mixtures thereof and in oils Under ordinary conditions of storage and use, these preparations contain a preservative to prevent the growth of microorganisms

Pharmaceutical forms suitable for use injectable include sterile aqueous solutions or dispersions; formulations that include sesame oil, peanut oil or aqueous propylene glycol; and sterile powders for preparation extemporaneous sterile injectable solutions or dispersions. In In all cases, the form must be sterile and must be fluid until the degree of easy injectability. It must be stable under manufacturing and storage conditions and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi

Truncated Tissue Factor compositions are they can formulate in a salt-shaped or shaped composition neutral Pharmaceutically acceptable salts, include salts addition acids (formed with the free amino groups of the protein) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or organic acids such as acetic, oxalic, tartaric, mandelic, and the like. Salts formed with free carboxyl groups can also be derived from inorganic bases such as, for example, sodium hydroxides, potassium, ammonium, calcium or ferric, and organic bases such as Isopropylamine, trimethylamine, histidine, procaine and the like.

The carrier can also be a solvent or a dispersion medium containing, for example, water, ethanol, polyol (for example glycerol, propylene glycol, and liquid polyethylene glycol, and similar), convenient mixtures thereof, and vegetable oils. Adequate fluidity can be maintained, for example, by use of a coating, such as lecithin, by maintenance of the particle size required in the case of dispersion and through the use of surfactants. The prevention of the action of microorganisms can be applied with respect to different substances antibacterial and antifungal, for example, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, and the like. In many cases, it will be it is preferable to include isotonic substances, for example, sugars or sodium chloride. Prolonged absorption of the compositions injectables can be applied by using compositions of substances that delay absorption, for example, monostearate of aluminum and jelly.

Sterile injectable solutions are prepared incorporating the active compounds in the amount required in the Suitable solvent with several of the other ingredients listed above, as required, followed by sterilization by filtration. Generally, dispersions are prepared by incorporating the various active ingredients sterilized in a sterile vehicle which contains the basic dispersion medium and the other ingredients required from those listed above. In case of dusts sterile for the preparation of sterile injectable solutions, Preferred methods of preparation are drying techniques vacuum, and lyophilization that produce a powder of the active ingredient plus some additional desired ingredient from the solution previously sterile filtered from it.

After formulation, the solutions are administered in a manner compatible with the formulation of the dose and in an amount that is therapeutically effective. The Formulations are easily administered in a variety of ways doses, such as the type of injectable solutions described previously, but release capsules can also be used of the drug and the like.

Convenient pharmaceutical compositions of according to the invention they will generally include an amount of Poor coagulation tissue factor mixed with a diluent or pharmaceutically acceptable excipient, such as aqueous solution sterile, to give a range of final concentrations, depending of the intended use. Preparation techniques are generally very known in the art as exemplified by Remington's Pharmaceutical Sciences, 16th Ed. Mack Publishing Company, 1980. It should be appreciated that endotoxin contamination should stay at a safe level, for example, less than 0.5 nanograms / milligram protein. Moreover, for the administration human, preparations should satisfy sterility, Pyrogenicity, general safety and purity standards as requires the FDA Office of Biological Standards (`` Food and Drug Administration '').

Therapeutically effective doses are easily determinable using an animal model, as shown in the studies detailed in this description. The animals Experiments that have solid tumors are often used to optimize the appropriate therapeutic doses before moving to A clinical environment These models are known to be very reliable to predict effective anticancer strategies. For example, the mice that have solid tumors, such as those used in Examples are widely used in preclinical tests. The inventors have used these mouse models accepted in the art to determine the working ranges of the Truncated Tissue Factor that give beneficial antitumor effects with minimal toxicity.

In addition to the compounds formulated for parenteral administration, such as intravenous injection or intramuscular, other forms are also taken into account pharmaceutically acceptable, for example, tablets or other solids for oral administration, delayed-release capsules, forms Liposomal and the like. Other pharmaceutical formulations too can be used, depending on the condition to be treated. By For example, topical formulations are suitable for treating pathological conditions such as dermatitis and psoriasis; Y Ophthalmic formulations for diabetic retinopathy.

As described in detail herein description, it is taken into account that certain benefits will be result of the manipulation of Factor factor constructions Poor coagulation tissue to provide a half-life Live longer. These techniques include, but are not limited to, the manipulation or modification of the Tissue Factor molecule same, and also the conjugation of constructions of Factor of Woven to inert carriers, such as different proteins or non-protein components, including immunoglobulins and Fc parts. These compositions are called Factor constructions here. Fabric with longer half-life. It will be understood that the half-life more Long is not the same as pharmaceutical compositions for use in `` slow release ''. Slow release formulations they are usually designed to give a constant drug level over an extended period. By increasing the half-life of a drug, such as a Tissue Factor construct according with the present invention, it is intended to result in a high plasma level after administration, and that these levels remain for a longer time, but these levels generally decay depending on the construction drug-kinetics. Even if currently not preferred, slow release formulations of the construction of Fabric Factor and combinations thereof of in no way are excluded from use in the present invention.

C2 Therapeutic equipment

The present invention also discloses therapeutic equipment comprising the constructions of Factor de Truncated fabric described in this description. This teams they will generally contain, in a convenient container medium, a pharmaceutically acceptable formulation of at least one Construction of Deficient Coagulation Tissue Factor of according to the invention. The equipment can also contain other pharmaceutically acceptable formulations, such as those contain components to target Factor constructions of truncated fabric; extra coagulation factors, particularly Factor VIIa; bispecific antibodies, T cells, or others functional components for use, for example, in induction of antigen; components for use in antigen suppression, such as a cyclosporine, if necessary; antibodies or immunotoxins of different antitumor site; and any one or more of a range of chemotherapeutic drugs.

Teams can have only one item container containing the Truncated Tissue Factor, with or without additional components, or may have different elements containers for each desired substance. Also considered equipment comprising the separate components necessary to make a bispecific coagulating ligand or immunotoxin. Some Preferred equipment of the present invention includes a Construction of poor coagulation tissue factor that is impaired in its ability to activate Factor VII, packaged in a device for use in combination with the co-administration of Exogenous factor VIIa. In these devices the tissue factor mutant and Factor VIIa may be precomplexed, either in a combination of molar equivalent, or with a component in excess of other; or each of the components of Fabric Factor and Factor VIIa of the equipment can be kept separately within different containers before administration to a patient. Other equipment Preferred include a poor coagulation tissue factor in combination with a `` classic '' chemotherapeutic substance. This It is by way of example the considerations that are applicable to the preparation of these tissue factor equipment and combinations of equipment in general.

When equipment components are provided in one or more liquid solutions, the liquid solution is a aqueous solution, being a sterile aqueous solution particularly preferential. However, equipment components can be provide as dry powders. When reagents or components they are provided as dry powder, the powder can be reconstituted by adding a convenient solvent. It is considered that the solvent can also be provided in another element container.

The container element of the equipment generally It shall include at least one bottle, test tube, flask, bottle, syringe or other container element, in which the Truncated Tissue Factor, and any other desired substance and, preferably, in suitable installments. When components are included additional, the team will also usually contain a second bottle or other container in which they are placed, enabling the administration of designated separate doses. The teams too they can comprise a second / third container elements for contain a sterile, pharmaceutically acceptable, or other regulator diluent

Teams can also contain an item whereby the Truncated Tissue Factor is administered to a animal or patient, for example, one or more needles or syringes, or even an eye dropper, pipette, or other of these devices, from which one can inject the formulation into the animal or apply in a desired area of the body. The equipment of the present invention they will also typically include an element to contain the jars, or similar, and another component, in narrow confinement for commercial sale, such as, for example, plastic containers injection molded or blow molded, in which they are placed and they retain the jars and other desired appliances.

D. Treatment D1 Prothrombotic vessels

The compositions to be used in this invention They are widely applicable to the treatment of any disease, such as a benign or malignant tumor, which has as a component of the disease `` prothrombotic vessels ''. These associated diseases with the vasculature more particularly include solid tumors, malignant, and also benign tumors, such as BPH. Nevertheless, the treatment of diabetic retinopathy, vascular restenosis, arteriovenous malformations (AVM), meningioma, hemangioma, neovascular glaucoma and psoriasis; and also angiofibrone, arthritis, atherosclerotic plaques, corneal graft neovascularization, hemophilic joints, hypertrophic scars, syndrome Osler-Weber, pyogenic granuloma, fibroplasia retrolental, scleroderma, trachoma, vascular adhesions, Synovitis, dermatitis and even endometriosis are also not excluded.

The present invention is based on the use of Tissue Factor or Tissue Factor constructs in combination with other substances, in which the construction of Tissue Factor or combination has enough thrombogenic activity to disturb the procoagulant environment within the specific associated vessels with the disease, such as those of a vascularized tumor, in the thrombosis direction The environment in vessels in normal tissues is fibrinolytic, while in the tumor vessels it is procoagulant, that is, it predisposes towards thrombosis. The changes procoagulants in tumor vessels are partly the result of a local release of endothelial cytokines that activate cells, IL-1 and TNFα. Interleukin 1 is secreted by most tumor cells and macrophages activated TNFα is secreted by host cells that have infiltrated the tumor, including activated lymphocytes, macrophages, NK cells and LAK cells.

Interleukin 1 and TNFα induce a variety of changes in the vascular endothelium, including the Overregulation of the Tissue Factor, the sub-regulation of plasminogen activators and overregulation of the inhibitor of plasminogen activators, PAI-1 (Nawroth and Stern, 1986; Nawroth et al., 1988). These effects are magnified additionally due to tumor derived factors (Murray and collaborators, 1991; Ogawa et al., 1990), possibly VEGF The collective result of these and other changes is that the endothelium becomes more able to support the formation of thrombi and less able to dissolve fibrin, producing a predisposition towards thrombosis.

Therefore, in the light of the phenomena scientists described above, the inventors take into account that when coagulation tissue factors are administered poor, have enough residual thrombogenic activity to tilt the balance of the coagulation cascade towards the thrombosis in the vessels that are usually of nature prothrombotic (figure 3). Although an understanding is not necessary mechanistic of scientific reasoning in order to practice the present invention, it will be understood that the above explanation is a mechanism by which the invention can work. East mechanism is based less on the specific location of the Tissue factors within the vessels of a vascularized tumor, in opposition to other vessels, but nevertheless it is surprising that a equal biodistribution of the Tissue Factor, if this occurs, it can lead to an uneven effect on coagulation within disease sites such as within solid tumors. Given as it is naturally an inherent property of the tumor to maintain a network of blood vessels and continue in the angiogenic process, is Obviously the blood vessels associated with the tumor cannot be so predisposed to thrombosis that spontaneously or easily they support coagulation, since coagulation would necessarily give as a result the arrest of oxygen and nutrients to the cells tumor and would cause the tumor to self-destruct. Evidently, This does not happen.

It will be readily appreciated that the present invention It has significant utility in the treatment of diseases, such as vascularized tumors, regardless of a understanding of the mechanisms by which one can induce specific coagulation in the vessels associated with the disease. However, the inventors further reason that another mechanism that underlines the possible surprising action of Tissue Factor constructions is that Tissue Factors selectively bind to certain vascular endothelial cells in preference to those in other tissues or body sites (figure 3). In accordance with the above, if the Fabric Factor Truncated selectively binds to the vascular endothelium of the tumor after the injection, this would put him in contact with a lipid surface and would promote the assembly of complexes of initiation of coagulation in the tumor vessels. Maybe due to the prothrombotic nature of tumor vessels, there is a increase in the local concentration of Factors VIIa, IXa, Xa, the tissue factor trajectory inhibitor (ITFT) or others molecules that interact with the Tissue Factor, thus encouraging location.

The methods can be used to test the Location of the Truncated Tissue Factor by labeling the Factor of Truncated tissue, injecting it into mice that have tumors, and determining if it is actually located within the vessels of the tumor. Although you have scientific interest, carry out these studies it is not necessary to practice the present invention, since the coagulation tissue factor construct administration Poor advantageously results in antitumor effects specific independent of the precise mechanism of action that Underlies this phenomenon.

The present uses of Factor molecules Poor coagulation tissue to promote coagulation in Prothrombotic blood vessels are different from previous uses proposed for tissue factor constructions, such as Truncated Tissue Factor in combination with Factor VIIa. I know have proposed the Truncated Tissue Factor and Factor VIIa for the combined use in the treatment of blood disorders, such as hemophilia (U.S. Patent Nos. 5,374,617; 5,504,064; and 5,504,067). U.S.A. Nos. 5,346,991 and 5,589,363 they also describe the use of mutants K165A and K166A to inhibit coagulation in the treatment of myocardial infarction, and provide recombinant DNA sequences and vectors for their production.

It will immediately be appreciated that the objectives of the previous methodology are in direct contrast with the vessels Prothrombotic blood targets of the present invention. The `` prothrombotic '' blood vessels are in a dynamic state that predisposes them to coagulation, but in which coagulation does not It occurs in the natural environment. This is exemplified by the blood vessels within a vascularized tumor categorized as prothrombotic, but in which the tumor maintains a supply of sufficient and necessary blood to support maintenance and tumor growth On the contrary, the target sites within of an individual with a blood disorder, they are by themselves nature significantly deficient in its ability to withstand coagulation The combined methodology of Factor de Truncated fabric and Factor VIIa directed primarily for use in hemophiliacs has been proposed for use in conjunction with the control of postoperative bleeding or severe trauma in which an injury external has prevented the coagulation process from being effective necessary. This in turn is different than the intention of the present invention

The most important use of the present invention is believes that it is related to the treatment of malignant tumors, vascularized However, in addition to various diseases and disorders numbered above, the invention is also intended for use in therapy of other benign growths. A particular example of this is benign prostatic hyperplasia (BPH), which can be treated according to particular doses and treatment regimens presented below. For him Benign prostatic hyperplasia treatment can also be combine with other treatments currently practiced in the technique. For example, the attack of immunotoxins to markers located within hyperplasia Benign prostatic, such as PSA.

D2 Cancer and treatment

The location of the Truncated Tissue Factor and specific coagulation is exploited more preferably for uses Therapeutics of Truncated Tissue Factor in the treatment of cancers and tumors These uses may use Fabric Factor truncated alone or in combination with chemotherapeutic substances and / or immunotoxins or coaguligands. The compositions and methods given to know about this invention are widely applicable to the treatment of any malignant tumor that has a vascular component. The Typical vascularized tumors are solid tumors, particularly carcinomas, which require a component vascular for the provision of oxygen and nutrients. Tumors exemplary solids that can be treated using the invention include, but are not limited to, carcinomas of the lung, chest, ovary, stomach, pancreas, larynx, esophagus, testicles, liver, parotid, bile ducts, colon, rectum, cervix, uterus, endometrium, kidney, bladder, prostate, thyroid, squamous cell carcinomas, adenocarcinomas, small cell carcinomas, melanomas, gliomas, neuroblastomas, and the like.

The present invention is intended for use in the treatment of any patient with a solid tumor. However, in what the present invention is particularly successful is in the treatment of solid tumors of moderate sizes or large, patients in these categories are likely to receive more significant benefits of treatments according to the methods and compositions disclosed in the present description. In general, the invention can be used to treat tumors of approximately 0.3-0.5 centimeters and more, although it is a better use of the invention to treat tumors greater than 0.5 centimeters in size Of the studies already conducted in models Acceptable animals, tumors are believed to be approximately 1.0 or approximately 1.2 centimeters represent the size of tumors solids that are most effectively attacked by the constructions of Tissue Factor of the present invention. Therefore, the patients with tumors between 1.0 and approximately 2.0 centimeters in size will be in the preferred treatment group of patients in relation to the present therapies of Factor de Tissue, although tumors up to and including tumors more Large ones found in humans can also be treated.

Although the present methods are not intended generally as a preventive or prophylactic treatment, the use of The invention is also not limited to the treatment of patients who have tumors of moderate or large sizes. There are many reasons under this aspect of the breadth of the invention. For example, a patient presenting with a moderate or larger primary tumor may also have several other metastatic tumors that considered small or even in the early stages of implantation of metastatic tumors. Since the constructions of Tissue Factor and combinations of the invention are administered. generally in a patient's systemic circulation, naturally they will have effects on secondary tumors, more small and metastatic, although this should not be the intention Primary treatment In addition, even in situations where the tumor mass as a whole is a single small tumor, certain benefits of antitumor effects will result from the use of those present treatments

The guide released above regarding to the most convenient patients for use in relation to the This invention is intended as teaching that certain patient profiles can help in the selection of patients who they can be treated by the present invention or that, perhaps, it is better to treat them using other treatment strategies anticancer However, the fact that one treatment or another most effective way is perceived to exist in relation to certain category of patients, in no way denies the usefulness basic of the present invention in relation to the treatments of All patients who have a vascularized tumor. Other Consideration is the fact that the initial assault on a tumor, provided by the tissue factor therapy of the present invention, can be less in any immediate and measurable effect, but it can predispose the tumor to other therapeutic treatments of so that subsequent treatment results in an effect Global synergistic or even lead to total remission or cure.

It is not believed that some type should be excluded particular tumor of the treatments using this invention. As the intent of therapy is to coagulate the vasculature of the tumor, and as the vasculature is substantially or entirely the same in all solid tumors, it will be understood that the present methodology is broad or entirely applicable to the treatment of all solid tumors, regardless of phenotype or particular genotype of the same tumor cells. However the type of tumor cells may be important for the use of the invention in combination with secondary therapeutic substances, particularly immunotoxins and / or cell coaguligands antitumor

Technicians with ordinary experience in the field will understand that certain types of tumors may be more docile to the induction of thrombosis and necrosis using the present invention. The phenomenon is observed in experimental animals, and can Present in human treatments. For example, it is known that the HT29 model tumor system is relatively difficult to coagulate; while the C1300 tumor model is generally more tame for induction of thrombosis and subsequent necrosis. These considerations will be taken into account to drive both the preclinical studies in experimental animals as in optimizing the doses for use in the treatment of any patient particular or group of patients.

As detailed earlier in the discussions concerning live quantitative studies, there are objectives actuals that can be used as guidelines in relation to preclinical tests before proceeding to clinical treatment. Without However, this is more a matter of cost effectiveness than of global utility, and is a mechanism to select compounds and most advantageous doses. With respect to its basic utility, any construction or combination thereof that results in a consistent detectable thrombosis and antitumor effects yet will define a useful invention. Thrombotic effects and necrotic between approximately 10% and approximately 40-50% of the blood vessels of the tumor and of the tumor tissues, observed up to about 50% and approximately 99% of these effects. It will also be understood that still in circumstances where the antitumor effects of construction of Fabric Factor and combinations are towards the lower end of this range, it may be that this therapy is equally or even more effective than all other known therapies in the context of the particular tumor targets. Unfortunately it is obvious to a doctor that certain tumors cannot be effectively treated to medium term or long term, but this does not deny the usefulness of the present therapy, particularly when it is as effective as others generally proposed strategies.

To design appropriate doses of Coagulation Tissue Factor constructs and combinations poor, can easily be extrapolated from animal studies described in the present description in order to arrive at doses suitable for clinical administration. To achieve this conversion, the mass of the substances administered would be considered per unit mass of the experimental animal, and then would be considered the differences in body surface area between the animal Experimental and the human patient. All these calculations are very known and routine to those skilled in the art. For example, taking the successful dose of 16 micrograms per mouse (body weight total of approximately 20 grams), and applying the calculation indicated above, the equivalent dose for use in a patient human would be about 2 milligrams. In accordance with Previous, using this information, the inventors take into account What useful doses of Deficient Coagulation Tissue Factor for its use in human administration would be between approximately 0.2 milligrams and approximately 200 milligrams of building Tissue Factor per patient. Notwithstanding this established range, it will be understood that, given the parameters and the detailed guide presented above, other variations will still be covered in the active or optimal ranges within the present invention.

The doses taken into account therefore they will generally be between about 0.2 milligrams and approximately 180 milligrams; between 0.5 and approximately 160 milligrams; between 1 and about 150 milligrams; between about 5 and about 125 milligrams; between about 10 and about 100 milligrams; between about 15 and about 80 milligrams; between about 20 and about 65 milligrams; between about 30 and about 50 milligrams; approximately 40 milligrams; or in any particular range using any of the doses given by way of example mentioned above or any intermediate value between the particular established ranges. Even if doses about 1 milligram, 2 milligrams, 3 milligrams, 4 milligrams and 5 milligrams are currently preferred, it will be understood that lower doses may be more suitable in combination with others substances, and that higher doses can still be tolerated, particularly given the fact that Factor substances Woven for use in the invention are not themselves cytotoxic. and even if there is an adverse side effect, it does not would necessarily result in coagulation that could not be counteracted by normal homeostatic mechanisms, which is believes that decreases opportunities for significant toxicity in healthy tissues

The intention of therapeutic regimens is generally produce maximum antitumor effects and at the same time keeping the dose below the levels associated with unacceptable toxicity. In addition to varying the same dose, the regimen administration can also be adapted to optimize the treatment strategy A treatment strategy currently Preferred is to administer between approximately 0.2 milligrams, and approximately 200 milligrams of the Factor construction Fabric or combination thereof approximately 3 times in approximately a period of 7 days. For example, the doses are they would give approximately day 1, day 3 or 4 and day 6 or 7. To administer the same particular doses, would be preferred provide a pharmaceutically acceptable composition to the patient systemically Intravenous injection is generally preferred, and the most preferred method is to use a continuous infusion during a period of time about 1 or 2 hours or so. But not It is required to determine these parameters before treatment using the present invention, it should be noted that detailed studies in the present description they result in at least some of thrombosis, specifically observed in the blood vessels of a solid tumor within approximately 30 minutes of the injection, and the tumor cells themselves begin to die inside about 3 to 4 hours. The extent is usually observed of tumor necrosis in the next approximately 24 hours, up to and including more than 90% of necrosis.

E. Combination therapies

The methods can be combined with any other method generally used in the treatment of the disease or particular disorder that the patient presents. For example in In relation to the treatment of solid tumors, the methods are they can use in combination with classic approaches, such as Surgery, radiotherapy and the like. As a therapeutic approach do not know that it is harmful in itself, or counteract the effectiveness of Tissue Factor therapy, is taken into account its combination with the present invention. When one or more are used substances in combination with Tissue Factor therapy, do not know requires that the combined results add up to the effects observed when each treatment is carried out separately, although this is obviously desirable, and there is no requirement particular so that the combined treatment exhibits effects synergistic, although this is certainly possible and advantageous.

In terms of surgery, any intervention Surgical can be performed in combination with the present invention. In relation to radiotherapy, it is taken into account any mechanism to induce DNA damage locally within tumor cells, such as γ irradiation, x-rays, ultraviolet irradiation, microwave and even electronic emissions and the like Targeted administration of radioisotopes at tumor cells are also taken into account, and this can be used in relation to a target antibody or other target elements or objectives. Cytokine therapy has also proven to be effective partner for combined therapeutic regimens. Can be employ several cytokines in these combined approaches. Examples of cytokines include IL-1α IL-1?, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-11, IL-12, IL-13, TGF-?, GM-CSF, M-CSF, G-CSF, TNFα, TNF?, LAF, TCGF, BCGF, TRF, BAF, BDG, MP, LIF, OSM, TMF, PDGF, IFN-?, IFN-?, IFN- γ. Cytokines are administered from according to standard regimes, consistent with indications clinics such as the patient's condition and relative toxicity of the cytokine.

E1. Chemotherapeutic combinations and treatment

In certain embodiments, the present invention shows that the antitumor activity of the Truncated Tissue Factor increases when administered in combination with a substance Chemotherapeutic The mechanisms by which drugs increase the antitumor activity of the Truncated Tissue Factor is not have defined precisely, but the inventors believe that the drug kills proliferating tumor cells creating necrotic areas that cause phagocytic cells to infiltrate the tumor. IL-1, TNFα and other cytokines released by the infiltrating cells then activate the vascular endothelium of the tumor making it more possible to support coagulation by the Factor of Truncated tissue, a generally weak thrombogen. The drug like this Increases the thrombotic action of Truncated Tissue Factor.

Another possibility for increased shares of the Tissue Factor and anticancer drugs is that the Factor of Truncated tissue induces thrombus formation in the vessels of the tumor, thus trapping the drug inside the tumor. Although the drug It is removed from the rest of the body, remains inside the tumor. The tumor cells are then exposed to a higher concentration of drug for a longer period of time. This entrapment of drug inside the tumor can also make it possible to reduce the dose of the drug, making the treatment safer as well as more cash.

Regardless of the mechanisms through which is the increased destruction of the tumor, the aspects of combined treatment have obvious utility in the treatment Effective of the disease. To use the present invention in combination with the administration of a substance chemotherapeutic, an animal would simply be administered a Coagulation Tissue Factor construction deficient in combination with the chemotherapeutic substance in a way effective to result in its combined antitumor actions inside the animal These substances would therefore be provided. in an effective amount and for an effective period of time to result in its combined presence within the vasculature of the tumor and its combined actions in the environment of tumor. To achieve this goal, the Tissue Factor and the substances Chemotherapeutics can be administered to the animal simultaneously, either in a single composition or as two different compositions using different routes of administration.

Alternatively, the Factor treatment Tissue may precede or follow the substance treatment Chemotherapy at intervals ranging from minutes to weeks. In embodiments where the chemotherapeutic factor and the Factor of Tissue were applied separately to the animal, usually would ensure that a significant period of time did not expire between time of each administration, so that the substance Chemotherapy and the composition of Tissue Factor still they would be able to exert a combined effect advantageously on the tumor In these cases, it is taken into account that there would be contact of the tumor with both substances within about 5 minutes up to about a week of each and, more preferably, within 12-72 hours of the other, being the most preferably a delay time of only about 12-48 hours In some situations, it can be desirable to extend the period of time for treatment significantly, where several days (2, 3, 4, 5, 6 or 7) or until several weeks (1, 2, 3, 4, 5, 6, 7 or 8) pass between respective administrations. It is also conceivable that more is desired of an administration of the Tissue Factor or the substance Chemotherapeutic To achieve tumor regression, both substances are administered in a combined effective amount to inhibit its growth regardless of the times for administration.

A variety of chemotherapeutic substances are intended for use in combined treatment methods described in the present description. The substances Chemotherapeutics taken into account by way of example include, for example, etoposide (VP-16), adriamycin, 5-fluorouracil (5FU), camptothecin, actinomycin-D, mitomycin C, cisplatin (CDDP) and to hydrogen peroxide. In certain embodiments, the use of etoposide has already been shown to be effective in the regression of the size of the tumor when administered in combination with the compositions of Truncated Tissue Factor of the present invention.

As those skilled in the art will understand, an adequate dose of chemotherapeutic substances generally It will be around those already used in clinical therapies where Chemotherapeutic substances are administered alone or in combination with other chemotherapeutic substances. By way of example only substances such as cisplatin can be used, and other DNA alkylating substances. Cisplatin has been used widely to treat cancer, with effective doses used in clinical applications of 20 mg / m2 for 5 days every three weeks for a total of three courses. Cisplatin is not absorbed orally and therefore should be applied by injection intravenously, subcutaneously, intratumorally, or intraperitoneally.

Other useful substances include compounds that interfere with DNA replication, mitosis and segregation chromosomal These chemotherapeutic compounds include adriamycin, also known as doxorubicin, etoposide, Verapamil, podophyllotoxin, and the like. Widely used in a clinical scenario for the treatment of neoplasms, these compounds are administered through bolus injections intravenously at doses ranging from 25-75 mg / m2 at 21-day intervals for adriamycin, up to 35-50 mg / m2 for ethoposide intravenously or double the intravenous dose orally.

Substances that disrupt synthesis and fidelity of polynucleotide precursors can also be use. Particularly useful are the substances that have suffered Extensive tests and are readily available. As such, it preferentially use substances such as 5-fluorouracil (5-FU) for tissue neoplastic, making this substance particularly useful for target neoplastic cells Although quite toxic, the 5-fluorouracil, is applicable in a wide range of carriers, including topical, however administration intravenously with doses ranging from 3 to 15 mg / kg / day is used Commonly.

Chemotherapeutic substances as example that are useful in relation to combined therapy is indicated in Table II. Each of the substances indicated in the they are by way of example and by no means limiting. The expert technician is referred to `` Remington's Pharmaceutical Sciences '' fifteenth edition, chapter 33, in particular pages 624-652. There will be some variation in dose necessarily depending on the state of the subject being trying. The person responsible for the administration, in In any case, it will determine the appropriate dose for the subject individual. Moreover, for human administration, the preparations must meet the standards of sterility, pyrogenicity, safety general and purity required by the Office of Biological Standards of the United States Food and Drug Administration of North America.

TABLE II Chemotherapeutic substances useful in disease neoplastic

       \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {l | l | l | l} \ hline
Class \ + Type of \ + Names not \ + Disease \\ \ + Substance
\ + Registered \ + \\ \ + \ + (other \ + \\ \ + \ + names) \ +
\\\ hline \ + \ + Mechloroetamina \ + Hodgkin's disease, lymphomas
\\ \ + \ + (HN2) \ + non-Hodgkin \\\ dddcline {3} {4}
\ + \ + Cyclophosphamide \ + Acute lymphocytic leukemia and \\ \ + \ +
Chronic ifosfamide \ +, Hodgkin's disease, \\ \ + \ + \ +
non-Hodgkin lymphomas, \\ \ + \ + \ + multiple myeloma, \\
  \ + \ + \ + neuroblastoma, sinus, ovary, \\ \ + Mustards \ + \ +
lung, Wilms tumor, Cervix, \\ \ + of \ + \ + testicles,
tissue sarcomas \\ \ + Nitrogen \ + \ + soft \\\ dddcline {3} {4}
  \ + \ + Melfalan (L- \ + Multiple myeloma, sinus, ovary \\ \ + \ +
sarcolysin) \ + \\\ dddcline {3} {4} \ + \ + \ + Lymphocytic leukemia
chronic, \\ \ + \ + Chlorambucil \ + primary macroglobulinemia, \\
 \ + \ + \ + Hodgkin's disease, lymphomas \\ \ + \ + \ + that don't
they are from Hodgkin \\\ dddcline {2} {4} \ + Ethylene- \ + Hematilmela- \ +
Ovary \\ \ + ore and \ + mine \ + \\ \ + Metilme- \ + \ +
\\\ dddcline {3} {4} Substances \ + sheets \ + Tiotepa \ + Bladder,
sine, ovary, \\\ dddcline {2} {4} alkylating agents \ + Sulfonate \ + \ + \\
 \ + de \ + Busulfan \ + Chronic granulocytic leukemia \\ \ +
alkyl \ + \ + \\\ dddcline {2} {4} \ + \ + \ + Hodgkin's disease,
Lymphomas \\ \ + \ + Carmustine \ + non-Hodgkin tumors
\\ \ + \ + (BCNU) \ + primary of the brain, myeloma \\ \ + \ +
\ + multiple, malignant melanoma \\\ dddcline {3} {4} \ + \ + \ +
Hodgkin's disease, lymphomas \\ \ + Nitrosou- \ + Lomustine \ +
that are not from Hodgkin, tumors \\ \ + reas \ + (CCNU) \ + del
primary brain, lung \\ \ + \ + \ + small cell
\\\ dddcline {3} {4} \ + \ + Semustina \ + Brain tumors
primary, \\ \ + \ + (methyl- CCNU) \ + stomach, colon
\\\ dddcline {3} {4} \ + \ + Streptozocin \ + Pancreatic Insulinoma
malignant, \\ \ + \ + (streptozoto- \ + malignant carcinoid \\ \ +
\ + cina) \ + \\ \ + Triazines \ + Dacarbazine \ + Malignant melanoma,
\\ \ + \ + disease (DTIC; \ + Hodgkin, tissue sarcomas \\
 \ + \ + dimethyltria- \ + soft \\ \ + \ + zenoimidazol- \ + \\ \ +
 \ + carboxamide) \ +
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
    

TABLE II (continuation)

       \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {l | l | l | l} \ hline
Class \ + Type of \ + Names not \ + Disease \\ \ + Substance
\ + Registered \ + \\ \ + \ + (other \ + \\ \ + \ + names) \ +
\\\ hline \ + \ + Methotrexate \ + Acute lymphocytic leukemia, \\
\ + Analogs \ + (ametopterin) \ + choriocarcinoma, mycosis \\
Antimetabo- \ + of Acid \ + \ + fungoides, sinus, head and \\ litos
 \ + Folic \ + \ + neck, lung, sarcoma \\ \ + \ + \ +
osteogenic \\\ dddcline {2} {4} \ + \ + Fluoracil \ + Sinus, colon,
stomach, \\ \ + \ + (5-fluoroura- \ + pancreas,
ovary, head and \\ \ + \ + cil; 5-FU) \ + neck,
urinary bladder, \\ \ + Analogs \ + Floxuridine \ + lesions of the
Premalignant skin \\ \ + de \ + (fluorodeso- \ + (topical) \\ \ +
Pyrimi- \ xiuridine; \ + \\ \ + dina \ + FUdR) \ +
\\\ dddcline {3} {4} \ + \ + Citarabine \ + Granulocytic leukemia
acute and \\ \ + \ + (cytosine \ + acute lymphocytic \\ \ + \ +
arabinoside) \ + \\\ hline \ + \ + Mercaptopurine \ + Leukemias
acute lymphocytic, \\ \ + \ + (6-mercaptopu- \ +
acute granulocytic and \\ \ + \ + rina; 6-MP) \ +
chronic granulocytic \\\ dddcline {2} {4} \ + Analogs \ + Thioguanine
 \ + Acute lymphocytic leukemias, \\ Purina \ +
(6-thioguanine; \ + acute granulocytic and \\ \ + e
\ + TG) \ + chronic granulocytic \\ \ + Inhibited- \ + \ +
\\\ dddcline {3} {4} \ + res \ + Pentostatin \ + Cell leukemia
hairy, \\ \ + Relationship \ + (2-deoxicofor- \ +
mycosis fungoides, leukemia \\ \ + swims \ + mycin) \ + lymphocytic
Chronic \\\ hline \ + \ + Vinblastine \ + Hodgkin's disease,
\\ \ + \ + (VLB) \ + non-Hodgkin lymphomas, breast, \\ \ +
\ + \ + testicles \\\ dddcline {3} {4} \ + Alcaloi- \ + \ + Leukemia
acute lymphocytic, \\ \ + des vinca \ + \ + neuroblastoma, tumor of
Wilms, \\ \ + \ + Vincristine \ + rhabdomyosarcoma, \\ disease
  \ + \ + \ + Hodgkin, lymphomas that are not \\ \ + \ + \ +
Hodgkin, cell lung \\ \ + \ + \ + small \\\ dddcline {2} {4}
 \ + \ + \ + Testicles, cell lung \\ \ + Epipode- \ +
Etoposide \ + small and other lungs, breast, \\ \ + filotoxi- \ +
Tertiposide \ + Hodgkin's disease, lymphomas \\ \ + nas \ + \ +
Non-Hodgkin leukemia \\ \ + \ + \ + acute granulocytic,
\\ \ + \ + \ + Kaposi sarcoma \\\ dddcline {2} {4} Products \ + \ +
\ + \\ natural \ + \ + Dactinomycin \ + Choriocarcinoma, tumor of
Wilms, \\ \ + \ + (actinomycin \ + rhabdomyosarcoma, testicles, \\
  \ + \ + D) \ + Kaposi's sarcoma \\\ dddcline {3} {4} \ + \ +
Daunorubicin \ + Acute granulocytic leukemia and \\ \ + \ +
(daunomycin; \ + acute lymphocytic \\ \ + \ + rubidomycin) \ +
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
    

TABLE II (continuation)

       \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {l | l | l | l} \ hline
Class \ + Type of \ + Names not \ + Disease \\ \ + Substance
\ + Registered \ + \\ \ + \ + (other \ + \\ \ + \ + names) \ +
\\\ hline Products \ + \ + \ + Soft tissue sarcomas, \\
natural \ + \ + \ + osteogenic and others; disease \\ (follow)
\ + Antibió- \ + Doxorubicina \ + of Hodgkin, lymphomas that are not \\
  \ + ticos \ + \ + of Hodgkin, acute leukemia, \\ \ + \ + \ +
breast, genitourinary, thyroid, \\ \ + \ + \ + lung, stomach,
neuroblastoma \\ \ + \ + \ + \\ \ + \ + \ + Testicles, head and
neck, \\ \ + \ + Bleomycin \ + skin, esophagus, lung and pathways \\
\ + \ + \ + genitourinary; \\ \ + \ + Hodgkin's disease,
non-lymphomas of \\ \ + \ + \ + Hodgkin \\\ hline \ + \ +
Plicamycin \ + Testis, hypercalcemia \\ \ + \ + (mitramycin)
\ + malignant \\\ dddcline {3} {4} \ + \ + Mitomycin \ + Stomach,
cervix, colon, breast, \\ \ + \ + (mitomycin C) \ + pancreas,
bladder, head and \\ \ + \ + \ + neck \\\ dddcline {2} {4} \ +
Enzymes \ + L-Asparaginase \ + Lymphocytic leukemia
acute \\\ dddcline {2} {4} \ + Modify- \ + \ + Cell leukemia
pilosas, \\ \ + dores of \ + \ + Kaposi's sarcoma, melanoma, \\
\ + Answers- \ + Interferon \ + carcinoid, renal cell, \\ \ + tas
 \ + alpha \ + ovary, bladder, lymphomas not \\ \ + Biological- \ +
\ + are from Hodgkin, mycosis \\ \ + cas \ + \ + fungoides, myeloma
multiple, \\ \ + \ + \ + chronic granulocytic leukemia \\\ hline
\ + Complexes \ + \ + Testicles, ovary, bladder, \\ de +
Cisplatin \ + head and neck, lung, \\ \ + Coordinates- \ +
(cis -DDP) \ + thyroid, cervix, endometrium, \\ \ + tion \ +
Carboplatin \ + neuroblastoma, sarcoma \\ \ + Platinum \ + \ +
osteogenic \\\ dddcline {2} {4} \ + Antrace- \ + Mitoxantrone \ +
Acute granulocytic leukemia, \\ Substances \ + nediona \ + \ +
breast \\\ dddcline {2} {4} several \ + Urea \ + \ + Leukemia
chronic granulocytic, \\ \ + Substitute- + Hydroxyurea \ +
polycythemia vera, thrombocytosis \\ \ + da \ + \ + esental, melanoma
malignant \\\ dddcline {2} {4} \ + Derivative \ + Procarbazine \ + \\ \ +
Methyl \ + (N-methylhydra- \ + Hodgkin's disease
\\ \ + hydrazine \ + zina, MHI) \ + \\ \ + \ + \ + \\ \ + Adreno- \ +
Mitotane \ + \\ \ + cortical \ + (or, p'-DDD) \ +
Adrenal cortex
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
    

TABLE II (continuation)

       \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {l | l | l | l} \ hline
Class \ + Type of \ + Names not \ + Disease \\ \ + Substance
\ + Registered \ + \\ \ + \ + (other \ + \\ \ + \ + names) \ +
\\\ hline \ + Suppressor \ + Aminogluteti- \ + Sinus \\ \ + \ + measure
\ + \\\ hline \ + Adreno- \ + Prednisone \ + Lymphocytic leukemia
acute and \\ \ + cortico- \ + (several other \ + chronic lymphomas that
they are not from \\ \ + steroi- \ + preparations \ + Hodgkin, disease
of Hodgkin, \\ \ + des \ + equivalents \ + sine \\ \ + \ +
available) \ + \\\ dddcline {2} {4} \ + \ + Hidroxiproges- \ + \\ \ +
 \ + terona \ + \\ \ + Progesti- \ + caproate \ + Endometrium, sine \\
  \ + na \ + Medroxiproges- \ + \\ Hormones and \ + \ + terona \ + \\
antagonis- \ + \ + Acetate of \ + \\ tas \ + \ + Megestrol \ +
\\\ dddcline {2} {4} \ + \ + Dietilestil- \ + \\ \ + \ + bestrol \ + \\
  \ + \ + Ethinyl \ + \\ \ + Estrogen \ + estradiol \ + Sinus,
prostate \\ \ + \ + (other \ + \\ \ + \ + preparations \ + \\ \ +
\ + available) \ + \\\ dddcline {2} {4} \ + Anties- \ + Tamoxifén \ +
Sinus \\ \ + troogen \ + \ + \\\ dddcline {2} {4} \ + \ + Testosterone
\ + Sinus \\ \ + Andróge- \ + propionate \ + \\ \ + us \ +
Fluoxymester- \ + \\ \ + \ + na (other \ + \\ \ + \ + preparations
\ + \\ \ + \ + available) \ + \\\ dddcline {2} {4} \ + Antian- \ +
Flutamide \ + Prostate \\ \ + drhogen \ + Leuprolide \ + Prostate
\\ \ + Analog \ + \ + \\ \ + of \ + \ + \\ \ + hormone \ + \ + \\ \ +
that \ + \ + \\ \ + releases \ + \ + \\ \ + Gonado- \ + \ + \\ \ + tropina
\ + \ +
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
    

E2 Combinations of immunotoxin and coaguligating and therapy

One or more Factor constructions can be used of poor coagulation tissue of the invention in combination with immunotoxins (ITs) and / or coaguligands in which the part target thereof (eg, antibody or ligand) is directed to a relatively specific marker of tumor cells, the Vasculature of the tumor or stroma of the tumor. In common with the Chemotherapeutic substances discussed above, it is possible that the use of a coagulation tissue factor construction poor in combination with a targeted toxic substance (IT) or coagulant (coaguligand) will result in substances different are directed against different objectives within the environment of the tumor This should lead to additive results, markedly greater than additives or even synergistic.

In relation to the preparation and use of by way of example immunotoxins and coaguligands, the following Descriptions of patent applications also complement the Present knowledge: U.S.A. with serial numbers 07 / 846,349; 08 / 295,868; 08 / 205,330; 08 / 350,212; 08 / 273,567; Y 08 / 482,369.

At least one link region of the second substances used in combination with Factor constructions The truncated fabric of the present invention will be a capable component of administering a toxin or a clotting substance to the region of the tumor, that is, capable of being located within a site of tumor location Since a somewhat wider distribution of the clotting substance will not be associated with side effects severe, there is a less strict requirement imposed on the item objective of coaguligands than with immunotoxins. The target substance can be directed to cell components of the tumor; components of the tumor vasculature; components that bind or are generally associated with tumor cells; components that bind or are generally associated with, the tumor vasculature; components of the extracellular matrix of tumor or stroma or those attached to them; and even types of cells found within the tumor vasculature.

With coaguligands, the burden of target restrictions, for example, that are imposed when They use immunotoxins. Therefore, achieve specific objectives means that coagulation is promoted in the vasculature of the tumor in relation to vasculature in non-localization sites of the tumor In this way, the specific objective of a coaguligand it is a functional term instead of a purely physical term in relationship with the properties of the biodistribution of the substance objective, and it is not unlikely that useful objectives cannot be completely restricted to the tumor, and that the ligands objectives that are effective in promoting specific coagulation of the tumor, however, can be found in other sites of the body after administration.

i. Objectives of tumor cells

The malignant cells that make up the tumor are they can attack using a ligand or a bispecific ligand that has a region capable of binding to a relatively specific marker of the tumor cell Toxins kill tumor cells and, in that link with the tumor cells will locate a substance coagulant associated with the tumor, and coagulation will be achieved specific.

Many of the so-called have been described `` tumor antigens '', any of which can be used as an objective in relation to the combined aspects of this invention. A large number of antigens are numbered below. associated with solid tumors by way of example. The preparation and use of antibodies against those antigens is very well within the technical expertise, and antibodies by way of example include gynecological tumor localization sites: OC 125; OC 133; IMO; Mo v1; Mo v2; 3C2; 4C7; ID_ {3}; DU-PAN-2; F 36/22; 4F7 / 7A10; OV-TL3; B72.3; DF_ {3}; 2C_ {8} / 2F_ {7}; MF 116; Mov18; CEA 11-H5; AC 19-9 (1116NS 19-9); H17-E2; 791T / 36; NDOG2; H317, 4D5, 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; HMFG2; 3.14.A3; sites of breast tumor location: DF3; NCRC-11; 3C6F9; MBE6; CLNH5; MAC 40/43; EMA; HMFG1 HFMG2; 3.15.C3; M3, M8, M24; M18; 67-D-11; D547Sp, D75P3, H222; Anti-EGF; LR-3; TA1; H59; 10-3D-2; HmAB1.2; MBR 1,2,3; 24 \ cdot17 \ cdot1; 24 \ cdot17 \ cdot2 (3E1.2); F36 / 22.M7 / 105; C11, G3, H7; B6 • 2; B1 • 1; Cam 17 \ cdot1; SM3; SM4; C-Mul (566); 4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, 5B8; OC 125; MO v2; DU-PAN-2; 4F7 / 7A10; DF_ {3}; B72 • 3; cccccCEA 11; H17-E2; 3 \ cdot14 \ cdotA3; FO23C5; of tumor localization sites colorectals: B72 • 3; (17-1A) 1083-17-1A; CO17-1A; ZCE-025; AB2; HT-29-15; 250-30.6; 44X14; A7; GA73 • 3; 791T / 36; 28A32; 28.19.8; X MMCO-791; DU-PAN-2; ID_ {3}; CEA 11-H5; 2C_ {8} / 2F_ {7}; CA-19-9 (1116NS 19-9); PR5C5; PR4D2; PR4D1; from melanoma sites 4 • 1; 8.2M_17, 96 · 5; 118.1, 132.2, (113 • 2); L 1, L 10, R 10 (R 19); I 12; K5; 6 \ cdot1; R24; 5 • 1; 225.28S; 465.12S; 9 \ cdot2 \ cdot27; F11; 376.96S; 465.12S; 15 • 75; 15 • 95; Mel-14; Mel-12; Me3-TB7; 225.28SD; 763.24TS; 705F6; 436910; M148; of gastrointestinal tumors: ID3; DU-PAN-2; OV-TL3; B72 • 3; CEA 11-H5; 3 \ cdot14 \ cdotA3; CCOLI; CA-19-9 (1116NS 19-9) and CA50; OC125; of lung tumors: 4D5 3H4, 7C2, 6E9, 2C4, 7F3, 2H11, 3E8, 5B8, 7D3, SB8; MO v2; B72 • 3; DU-PAN-2; CEA 11-H5; MUC 8-22; MUC 2-63; MUC 2-39; MUC 7-39; and of tumors miscellaneous: PAb 240; PAb 246; PAb 1801; ERIC \ cdot1; M148; FMH25; 6 \ cdot1; CA1; 3F8; 4F7 / 7A10; 2C_ {8} / 2F_ {7}; CEA 11-H5.

Another means of defining a dirigible tumor is in terms of the tumor cell characteristics itself, instead of describing the biochemical properties of an expressed antigen by the cell. In accordance with the above, the skilled technician will refers to the ATCC catalog for the purpose of exemplifying lines of human tumor cells that are available to the public (from ATCC catalog). Example cell lines include J82; RT4; ScaBER; T24; TCCSUP; 5637; SK-N-MC; SK-N-SH; SW 1088; SW 1783; U-87 MG; U-118 MG; U-138 MG; U-373 MG; Y79; BT-20; BT-474; MCF7; MDA-MB-134-VI; MDA-MD-157; MDA-MB-175-VII; MDA-MB-361; SK-BR-3; C-33 A; HT-3; ME-180; MS751; SiHa; JEG-3; Caco-2; HT-29; SK-CO-1; HuTu 80; A-253; FaDu; A-498; A-704; Caki-1; Caki-2; SK-NEP-1; SW 839; SK-HEP-1; A-427; Calu-1; Calu-3; Calu-6; SK-LU-1; SK-MONTH-1; SW 900; EB1; EB2; P3HR-1; HT-144; Malme-3M; RPMI-7951; SK-MEL-1; SK-MEL-2; SK-MEL-3; SK-MEL-5; SK-MEL-24; SK-MEL-28; SK-MEL-31; Caov-3; Caov-4; SK-OV-3; SW 626; Capan-1; Capan-2; DU 145; A-204; Saos-2; SK-ES-1; SK-LMS-1; SW 684; SW872; SW982; SW1353; U-2 OS; Malme-3; KATO III; Cate-1B; Tera-1; Tera-2; SW579; AN3 CA; HEC-1-A; HEC-1-B; SK-UT-1; SK-UT-1B; SW 954; SW 962; NCI-H69; NCI-H128; BT-483; BT-549; DU4475; HBL-100; Hs 578Bst; Hs 578T; MDA-MB-330; MDA-MB-415; MDA-MB-435S; MDA-MB-436; MDA-MB-453; MDA-MB-468; T-47D; Hs766T; Hs 746T; Hs 695T; Hs 683; Hs 294T; Hs 602; JAR; Hs 445; Hs 700T; H4; Hs 696; Hs913T; Hs 729; FHs 738Lu; FHs 173We; FHs 738 B1; NIH: 0VCAR-3; Hs 67; RD-ES; ChaGo K-1; WERI-Rb-1; NCI-H446; NCI-H209; NCI-H146; NCI-H441; NCI-H82; H9; NCI-H460; NCI-H596; NCI-H676B; NCI-H345; NCI-H820; NCI-H520; NCI-H661; NCI-H510A; D283 Med; Daoy; D341 Med; AML-193 and MV4-11.

You can consult the ATCC catalog of any later year to identify other suitable cell lines. Also, if a particular cell type is desired, the means for get these cells, and / or their instant available source, will be well known to those skilled in the particular art. An analysis of scientific literature will easily reveal a choice suitable for any type of desired tumor cell that It is going to attack.

(a) Antitumor cell antibodies

A direct element to recognize a goal tumor antigen is through the use of an antibody that has a binding affinity for the particular antigen. They know a large number of antibodies directed against antigens of solid tumors Certain useful antitumor antibodies are mentioned above. However, as experts in the technique may quickly realize certain antibodies mentioned will not have the appropriate biochemical properties, or may not have enough tumor specificity, to be useful therapeutically An example is MUC8-22 that recognizes a cytoplasmic antigen. Antibodies such as these they will generally be useful only in research environments, such as in model systems or analysis tests.

Generally speaking, antibodies useful in these aspects of the present invention will preferably recognize antigens that are accessible on the cell surface and that are preferentially, or specifically, expressed by cells Tumor These antibodies will preferably also exhibit high affinity properties, such as displaying a K d of <200 nM, and preferably, <100 nM, and will not show significant reactivity with vital normal tissues, such as one or more selected tissues of the heart, kidney, brain, liver, bone marrow, colon, breast, prostate, thyroid, gallbladder, lung, adrenal, muscle, nerve fibers, pancreas, skin, or other vital organs or tissues in the human body. The tissues `` vital '' that are more important for the purposes of this invention, from the point of view of low reactivity, include heart, kidney, central and peripheral nervous system tissues and liver. The term `` significant reactivity '', as used in the This description refers to an antibody or fragment of antibody, which, when applied to particular tissue under Convenient conditions for immunohistochemistry, you will get either no spotting or despicable spotting with only few positive cells scattered among a field of most cells negative

Particularly promising antibodies intended for use in the present invention are those that have high selectivity for the solid tumor. For example, antibodies that bind with TAG 72 and the HER-2 proto-oncogenic protein, which are selectively found on the surfaces of many breast, lung and colorectal cancers (Thor et al., 1986; Colcher et al., 1987; Shepard et al., 1991); MOv18 and OV-TL3 and antibodies that bind to the mucin core protein of milk and the human milk fat globule (Miotti et al. 1985; Burchell et al. 1983); and antibody 9.2.27 that binds to the high M r melanoma antigens (Reisfeld et al., 1982). Other useful antibodies are those against the protein that binds to folate, which is known to be expressed homogeneously in almost all ovarian carcinomas; those against the erb family of oncogens that are overexpressed in squamous cell carcinomas and most gliomas; and other antibodies known to be subjects of continuous preclinical and clinical evaluation.

Antibodies B3, KSI / 4, CC49, 260F9, XMMCO-791, D612 and SM3 are believed to be particularly suitable for use in clinical embodiments, following the standard preclinical testing routine practiced in the technique. B3 (U.S. Patent No. 5,242,813; Brinkmann and collaborators, 1991) has the access number ATCC HB 10573; KS1 / 4 It can be done as described in U.S. Pat. 4,975,369; Y D612 (U.S. Patent No. 5,183,756) has the access number of ATCC, HB 9796.

Other means to define the associated objectives to the tumor are in terms of the cell characteristics of the tumor, instead of describing the biochemical properties of a antigen expressed by the cell. In accordance with the above the inventors take into account that any antibody that binds preferentially a tumor cell can be used as the target component of an immunotoxin or coaguligating. The link of preferential tumor cell again is based on the antibody that exhibits high affinity for the tumor cell and it has no significant reactivity with normal cells or tissues vital, as defined above.

The invention also discloses various means. to generate an antibody for use in coagulation methods addressed described in the present description. To generate a tumor cell specific antibody, an animal would be immunized with a composition comprising a tumor cell antigen and, as described more fully below, a resulting antibody with adequate specificity. The composition immunizer may contain a purified preparation, or partially purified, of any of the aforementioned antigens previously; a composition, such as a preparation of membrane, enriched for any of the aforementioned antigens previously; any of the cells mentioned in the list previous; or a mixture or population of cells that include any of the cell types mentioned above.

Of course, regardless of the source of the antibody, in the practice of the invention in human treatment, is preferable to ensure in advance that the target tumor clinically express the finally selected antigen. This is achieved through a fairly direct essay, which involves testing antigenically a sample of tumor tissue, for example, a surgical biopsy, or maybe test the hidden circulation of the antigen. This can easily be done in a test of immunological analysis such as an ELISA assay (immunosorbent of binding enzyme), where the affinity of the antibody binding to Starting from a hybridoma `` bank '' is tested to determine its reactivity against the tumor. The antibodies that demonstrate Appropriate tumor selectivity and affinity are selected for preparation of bispecific antibodies herein invention.

Due to the well-known reactivity phenomenon crossed, it is taken into account that useful antibodies can be result of immunization protocols in which antigens originally employed were derived from an animal, such as a mouse or a primate, in addition to those in which they were obtained original antigens from a human cell. When used antigens of human origin, can be obtained from a cell line of human tumor, or they can be prepared by obtaining a sample biological of a particular patient in question. No doubt be they know methods for the development of antibodies that are made `` to customer measure '' for the patient's tumor (Stevenson and collaborators, 1990) and are taken into account for their use in relation With this invention.

(b) Other tumor cell targets and ligands of Union

In addition to the use of antibodies, they can be used other ligands to direct a coagulant substance to a site of tumor location ligand to a tumor cell antigen. For tumor antigens that have overexpressed receptors (estrogen receptor, EGF receptor), or mutant receptors, will could use the corresponding ligands as substances objective.

In a manner analogous to receptor ligands of endothelial cells, there may be many components that are specifically or preferentially bind to cells Tumor For example, if a tumor antigen is a receptor overexpressed, the tumor cell can be coated with a ligand Live specific. It seems that the ligand could then go with an antibody against the ligand, or with a form thereof receiver. Specific examples of this type of target substances are antibodies against TIE-1 or ligands TIE-2, antibodies against platelet factor 4, and leukocyte adhesion binding protein.

ii. Other target diseases

In other embodiments, the tissue factors in combination with immunotoxins or coaguligands that bind to a target molecule that is expressed specifically or preferentially at a disease site other than A tumor.

The target molecules by way of example associated with other diseased cells include, for example, leukocyte adhesion molecules, which are associated with psoriasis; FGF, which is associated with proliferative diabetic retinopathy; factor 4 of platelet, which is associated with the activated endothelium of several diseases; and VEGF, which is associated with proliferative disease vascular. It is believed that an animal or patient that has any of the above diseases would benefit from induction specific coagulation at the site of the disease and optionally of the administration of directed toxin.

The diseases that are known to have a common angiodependent pathology, as described in Klagsburn and Folkman (1990), can also be treated as described in the present description In particular, a cell-directed ligand Vascular endothelial or a stromal directed ligand will be used to achieve coagulation at the disease site. The tratment of BPH, diabetic retinopathy, vascular restenosis, adhesions vascular, AVM, meningioma, hemangioma, neovascular glaucoma, rheumatoid arthritis and psoriasis are particularly taken into account at this time.

iii. Vasculature cell targets associated with the disease

Vasculature cells are destined as objectives for use in the present invention. In these cases, at least one immunotoxin binding region or coaguligating will be able to link to a preferentially accessible bookmark expressed by associated vasculature endothelial cells With the disease The exploitation of vascular markers is possible due to the proximity of endothelial cells vascular to the area of the disease and to the products of the local aberrant physiological processes. For example, the cells Vascular endothelial tumors are exposed to tumor cells and to tumor-derived products that change the phenotypic profile of endothelial cells

It is known that tumor cells make tumor-derived products, such as lymphokines, monochines, colony stimulating factors, growth factors and factors angiogenic, which act on vascular endothelial cells nearby (Kandel et al., 1991; Folkman et al., 1985a, 1985b) and cytokines (Burrows et al., 1991; Ruco and collaborators, 1990; Borden et al., 1990). The products Tumors bind endothelial cells and serve to induce selectively the expression of certain molecules. In these molecules induced that can be attacked using the specific toxin of the tumor endothelium and / or coagulant administration disclosed by certain aspects of the present invention. The cells vascular endothelial tumors proliferate at a rate of 30 times greater than those in miscellaneous normal tissues (Denekamp and collaborators, 1982), suggesting that the determinants linked to proliferation would also serve as markers for cells vascular endothelial tumors.

In certain embodiments of the invention, the target component of immunotoxins or coaguligands will be a component that has a relatively high degree of specificity for the vasculature of the tumor. These objective components can be define as components that bind to molecules expressed in the tumor endothelium, but they have little or no expression in the surface of normal endothelial cells. This specificity can be assessed by standard immunostaining procedures of tissue sections, which are routine for experts in the technique In terms of coaguligands, it is generally proposed that the molecules that are going to attack using ligands bispecific or antibodies of this invention will be those that express in the vasculature of the tumor at a higher level than above normal endothelial cells.

(a) Vascular endothelial cell markers in disease

Molecules that are known to preferentially express themselves on the surface of vascular endothelial cells at a disease site or environment are referred to herein as `` vascular endothelial cell markers associated with natural disease. '' This term is used for simplicity to refer to endothelial cell components that are expressed in diseases related to increased or inappropriate angiogenesis or endothelial cell proliferation. A particular example is the components of tumor endothelial cells that are expressed in situ in response to tumor derived factors. These components are also called `` naturally induced tumor endothelial cell markers. ''

Both endothelial growth factor vascular / vascular permeability factor as the components of the FGF family (fibroblast growth factor) are concentrated in the vasculature of the tumor. The corresponding receivers, so both, provide a potential target for attack on the Vasculature of the tumor For example it is known that the receptors of the vascular endothelial growth factor are overregulated in the tumor endothelial cells, unlike cells endothelial in normal tissues, both in rodents and in the man (Thieme et al., 1995). Possibly, this is a consequence of hypoxia - -a characteristic of tumor microenvironment (Leith et al., 1992). The fibroblast growth factor receptors are also overregulate 3 times in endothelial cells exposed to hypoxia, and are thought to be overregulated in tumors (Bicknell and Harris et al., 1992).

The TGF receptor (endoglin) (factor of transformant growth)? on endothelial cells is overregulates division cells, providing another objective. One of the present inventors found that endoglin it is overregulated in activated and crop division HUVEC, and strongly expresses in human tissues on endothelial cells in neovascularization sites, including a wide range of tumors solid and fetal placenta. In contrast, endothelial cells in most of the non-malignant miscellaneous tissues of adults, including preneoplastic lesions, they contain little or no endogline Importantly, it is believed that the expression of endoglin correlates with neoplastic progression in the breast, as shown by benign fibroadenomas and early carcinomas that bind to low levels of TEC-4 antibodies and TEC-11, and late stage intraductal carcinomas and invasive carcinomas that bind to high levels of these antibodies

Other endothelial cell markers Vascular associated with natural diseases include SIT, VCAM-1, P-selectin, E-selectin (ELAM-1), α V β 3 integrin, pleitropin and endosialin, each of which can be attacked using the invention.

(b) Vascular endothelial markers inducible by cytokine

Due to the nature of the processes of disease, which often result in dysfunction located inside the body, there are methods available to manipulate the site of the disease while leaving other tissues relatively unaffected. This is particularly true in malignant and benign tumors, which exist as entities different within the body of an animal. For example, the environment of the tumor can be manipulated to create additional markers that are specific for vascular endothelial cells of the tumor. These methods generally mimic those presented naturally in solid tumors, and also involve the local production of pointing substances, such as factors of growth or cytokines, which induce the specific expression of certain molecules on the surface of endothelial cells vascular nearby.

The group of molecules that can be induced artificially to be expressed on the surface of the cells vascular endothelials in a tumor disease or environment are they call in the present description `` cell markers inducible endothelial, '' or specifically, `` markers of inducible tumor endothelial cells. '' This term is used to refer to artificially induced markers, it is that is, they are induced as a result of manipulation by the hand of the man, instead of those that are induced as part of a disease or tumor development process in an animal. The term `` marker inducible '', as defined above, is chosen by simple reference in the context of the present application, regardless of the fact that `` natural markers '' they are also induced, for example, by substances derived from tumor.

Thus, although it is not required for practice the invention, there are techniques available for obtaining selective targets of vascular endothelial antigens on the surface of the vasculature associated with disease that, if may be used together with the invention. These techniques involve the manipulation of antigenic expression, or the presentation on the cell surface, so that a target antigen or becomes available on the surface of the vasculature associated with the disease and is not expressed or of another mode becomes accessible or available for link, or at least in a lesser degree, on the surface of the normal endothelium.

Markers of the tumor endothelium can be induce by tumor-derived cytokines (Burrows et al., 1991; Ruco et al., 1990) and by angiogenic factors (Mignatti et al., 1991). Examples of markers for cell surface that can be specifically induced in the tumor endothelium and then attack using a coagulant ligand Bispecific, as disclosed by the invention, include mentioned in Table III (Bevilacqua et al., 1987; Dustin et al., 1986; Osborn et al., 1989; Collins and collaborators, 1984).

Table III also shows the mechanisms for induction of the proposed markers; the `` inductive cytokine '' or `` intermediate cytokine '', such as the IL-1 and IFN-?; and the kind of leukocyte cell and cytokine activation molecule associated, whose objective will result in the release of the cytokine In the induction of a specific marker, generally an antibody `` that induces cytokines '' or `` that induces antigen''. The antibody will selectively induce the release of adequate cytokine at the tumor site, thereby inducing selectively the expression of the desired target antigen by vascular endothelial cells. This bispecific antibody crosslinked tumor cell cells and leukocytes that produce cytokine, thereby activating leukocytes to that release the cytokine.

The preparation and use of bispecific antibodies such as these are based in part on the fact that the cross-linking of antibodies that recognize CD3, CD14, CD16 and CD28 previously it has been shown that they obtain cytokine production selectively after crosslinking with the second antigen (Qian et al., 1991). In the context of this invention, since only cell-cross-linked leukocytes Tumor will be successfully activated to release the cytokine, the Cytokine release will be restricted to the tumor site. Of this mode, the desired marker expression, such as the E-selectin, will be limited similarly to endothelium of the vasculature of the tumor.

TABLE III Possible vascular goals inducible

       \ catcode` \ # = 12 \ nobreak \ centering \ small \ begin {tabular} {| l | l | l | l | l | l |} \ hline
 Molecules \ + Acronym \ + subtypes / alias \ + Cytokines of \ +
Leukocytes that \ + inducible \\ molecules of \ + \ + (family
 \ + induction \ + produce these \ + leukocyte which, \\ cells \ +
\ + molecular) \ + \ + cytokines \ + when cross-linking \\
endothelial \ + \ + \ + \ + \ + by antibodies \\ \ + \ + \ + \ +
 \ + monoclonal, \\ \ + \ + \ + \ + \ + activate cells \\
\ + \ + \ + \ + \ + to produce \\ \ + \ + \ + \ + \ + cytokines
\\\ hline \ + ELAM-1 \ + - - \ + molecule
IL-1, TNF-a, (TNF \ + monocytes \ +
CD14 \\\ dddcline {5} {6} adhesion of \ + \ + (Selectina) \ +
- \ beta) (Endotoxin \ + macrophages \ + CD14 \\\ dddcline {5} {6}
leukocyte-endothelial-1 \ +
E-selectin \ + \ + bacterial) \ + primed cells
\ + FcR for IgE \\\ hline \ + VCAM-1 molecule
\ + \ + Molecule (Endotoxin \ + monocytes \ + CD14
\\\ dddcline {5} {6} adhesion \ + \ + cell adhesion \ + bacterial)
IL-1, \ + macrophages \ + CD14 \\\ dddcline {5} {6}
vascular of the inducible \ + \ + 110 \ + TNF-a \ +
primed cells \ + FcR for IgE \\\ dddcline {4} {6}
cell-1 \ + \ + (INCAM-110) \ +
TNF-?, IL-4 \ + T cells +
CD2, CD3, CD28 \\ \ + \ + (Family of \ + \ + helpers \ +
\\\ dddcline {4} {6} \ + \ + immunoglobulin) \ + TNF \ + NK cells
\ + FcR for IgG (CD16) \\\ hline \ + molecule
ICAM-1 \ + - - \ + IL-1,
TNF-a \ + monocytes \ + CD14 \\\ dddcline {5} {6}
adhesion \ + \ + (Family of \ + (Endotoxin \ + macrophages \ + CD15
\\\ dddcline {5} {6} intercellular-1 \ + \ +
immunoglobulin) \ + bacterial) \ + primed cells \ + FcR for
IgE \\\ dddcline {4} {6} \ + \ + \ + TNF- \ beta, IFNg
 \ + T cells + CD2, CD3, CD28 \\ \ + \ + \ + \ + helpers \ +
\\\ hline \ + \ + \ + \ + NK cells \ + FcR for IgG (CD16)
\\\ hline Substance for the \ + Substance \ + Substance
MEL-14 \ + IL-1, TNFa \ + monocytes
\ + CD14 \\\ dddcline {5} {6} adhe- \ + molecule
LAM-1 \ + (mouse) \ + (Endotoxin \ + macrophages \ +
CD14 \\\ dddcline {5} {6} leukocyte sion 1 \ + \ + \ + bacterial)
\ + primed cells \ + FcR for IgE \\\ hline \ + MHC antigen
Class \ + HLA-DR \ + IFN-g \ +
T cells + CD2, CD3, CD28 \\ complex of \ + II \ +
HLA-DP \ hfill -Human \ + \ + helpers \ +
\\ histocompatibi- \ + \ + HLA - DQ \ + \ + \ + \\
Majority \ + \ + \ + \ + \ + \\ class II \ + \ + IA
 \ hfill -Mouse \ + \ + \ + \\ \ + \ + IE \ + \ + \ +
\\\ dddcline {5} {6} \ + \ + \ + \ + NK cells \ + FcR for IgG
(CD16)
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
    

It is important to note that, of the markers possible inducible mentioned in Table III, the E-selectin, and MHC Class II antigens, such like HLA-DR, HLA-DP and HLA-DQ (Collins et al., 1984), are for much the most preferred objectives for use in relation to clinical realizations The other adhesion molecules of the Table III seem to express themselves in varying degrees in normal tissues, usually in lymphoid organs or endothelium, making its target perhaps suitable only in animal models or in cases where its expression on normal tissues can be inhibited without effects significant secondary. It is preferred to direct E-selectin or an MHC Class II antigen, since the expression of these antigens will probably be the most direct for selectively promote in tumor associated endothelium.

E-selectin

The objective of an antigen that is not expressed on normal endothelial surfaces is the most direct form of induction methods E-selectin is a adhesion molecule that is not expressed in endothelial vasculature normal or in other types of human cells. (Cotran and collaborators, 1986), but it can be induced on the surface of cells endothelial through the action of cytokines such as IL-1, TNF, lymphotoxin and bacterial endotoxin (Bevilacqua et al., 1987). It is not induced by IFN-? (Wu et al., 1990). The E-selectin expression can be induced from this selectively mode in tumor endothelium through the selective administration of a cytokine, or by the use of a composition that causes the selective release of these cytokines in the tumor environment.

Bispecific antibodies are an example of a composition capable of causing the selective release of one or more of the above or other suitable cytokines at the site of tumor location, but nowhere else in the body. These Bispecific antibodies are called in the present description. `` antibodies that induce antigen '' and are, of course, distinct of any bispecific antibody of the invention having white components and coagulants. The antibodies that induce antigen are designed to cross-link cytokine effector cells, such as monocyte / macrophage lineage cells, T cells and / or NK cells or primed cells, with mass tumor cells solid tumor target. This crosslinking would effect a release of cytokine that is located at the cross-linking site, is Say the tumor.

Antibodies effective to induce antigen recognize a tumor cell surface antigen, on the one hand, and on the other hand, they recognize an antigen `` that activates the cytokine '' on the surface of a selected type of leukocyte cell. The term antigen `` that activates the cytokine '' is used to refer to to any of the known molecules on the surfaces of the leukocytes that, when linked by an effector molecule, such as an antibody or fragment thereof or a substance that is naturally presents or a synthetic analogue thereof, either a soluble factor or a membrane bound counter receptor or other cell, promotes the release of a cytokine through the cell of leukocyte. Examples of molecules that activate the cytokine include CD14 (the LPS receiver) and FcR for IgE, which will activate the release of IL-1 and TNFα; and CD16, CD2 or CD3 or CD28, which activate the release of IFNγ and TNFβ, respectively.

As soon as it enters the bloodstream of an animal that has a tumor, this bispecific antibody that induces antigen will bind to tumor cells within the tumor, will crosslink those tumor cells with effector cells, by example, monocytes / macrophages, which have infiltrated the tumor, and after that they will effect the selective release of cytokine inside the tumor However, importantly without the tumor and leukocyte cross-linking, the antibody that induces antigen will not effect cytokine release. In this way, no cytokine release will occur in parts of the body removed from the tumor and therefore the expression of cytokine-induced molecules, for example, E-selectin, will be presented only within the tumor endothelium

Many `` activating antigens of Useful cytokines, which, when cross-linked with antibody suitable bispecific, will result in the release of cytokines by the reticulated leukocyte. The goal generally Preferred for this purpose is CD14, which is above the surface of monocytes and macrophages. When CD14 is crosslinked stimulates monocytes / macrophages to release IL-1 (Schutt et al., 1988; Chen and collaborators, 1990), and possibly other cytokines, which, at in turn, they stimulate the appearance of E-selectin on the nearby vasculature. Other possible objectives for crosslinking in relation to the induction of E-selectin and addressing include FcR for IgE, found in cells barley FcR for IgG (CD16), found in NK cells; as well as CD2, CD3 or CD28, found on the surfaces of T cells. these, it is preferred to target CD14 due to the prevalence relative infiltration of monocyte / macrophage of solid tumors in opposition to the other types of leukocyte cells.

In an induction embodiment by way of example, an animal that has a solid tumor is injected with bispecific antibody (Fab'-Fab ') anti-CD14 / antitumor (such as antibody anti-CEA, 9.2.27 against melanoma antigens Mr high OV-TL3 or MOv18 antibodies against antigens associated with the ovaries). The antibody is located in the tumor, by virtue of its tumor binding activity, and then active monocytes and macrophages in the tumor by crosslinking their CD14 antigens (Schutt et al., 1988; Chen et al., 1990). The activated monocytes / macrophages have tumoricidal activity (Palleroni et al., 1991) and release IL-1 and TNF that rapidly induces E-selectin antigens on vascular endothelial cells of the tumor (Bevilacqua and collaborators, 1987; Pober et al., 1991).

MHC Class II antigens

The second preferred group of inducible markers intended for use in the present invention are the MHC Class II antigens (Collins et al., 1984), which include HLA-DR, HLA-DP and HLA-DQ. Class II antigens are expressed on vascular endothelial cells in most normal tissues in several species, including man. In vitro studies (Collins et al., 1984; Daar et al., 1984; O'Connell et al., 1990) and in vivo (Groenewegen et al., 1985) have shown that the expression of Class II antigens by vascular endothelial cells requires the continuous presence of IFN-γ, which is made by T H1 cells and, to a lesser extent, by NK cells and CD8 + T cells.

MHC Class II antigens are not exclusive to vascular endothelial cells, and also express themselves constitutively in B cells, activated T cells, cells monocyte / macrophage lineage and in certain epithelial cells, both in mice (Hammerling, 1976) as in man (Daar et al., 1984). Due to the expression of MHC Class II antigens on `` normal '' endothelium, its goal is not as direct as the E-selectin However, induction and direction MHC Class II antigens are made possible by using them together with an immunosuppressant, such as cyclosporine A (CsA), which has the ability to effectively inhibit the expression of molecules of Class II in normal tissues (Groenewegen et al., 1985). The cyclosporine A acts by preventing the activation of T cells and cells NK (Groenewegen et al., 1985; DeFranco, 1991), reducing by this the basal levels of IFN-? below those necessary to maintain the expression of Class II in the endothelium

There are several other cyclosporins related to cyclosporine A, including cyclosporins A, B, C, D, G, and similar, which also have immunosuppressive action and probably demonstrate an ability to suppress Class II expression. Other substances that may be similarly useful include FK506 and rapamycin

Thus, the practice of induction of MHC Class II and the conduct of attack requires a pretreatment of animal that has a tumor with one dose of cyclosporine A or another Class II immunosuppressive substance that is effective for suppress the expression of Class II. In case of cyclosporine A, this is typically of the order of about 10 to approximately 30 mg / kg body weight. As soon as it is deleted in normal tissues, Class II antigens can also be selectively induce in the tumor endothelium, again through of the use of a bispecific antibody.

In this case, the bispecific antibody that induces the antigen will have specificity for a cell marker tumor and for an activation antigen found in the surface of an effector cell that is capable of inducing the IFN-γ production. These effector cells they will generally be helper T cells (TH) or cells natural eliminators (NK). In these embodiments, it is necessary that T cells, or NK cells if CD16 is used, are present in the tumor to produce the intermediate cytokine in which the Class II antigen expression is achieved using IFN-?, But not achieved with other cytokines. Thus, for the practice of this aspect of the invention, you will want to select CD2, CD3, CD28, or more preferably CD28, as the cytokine activating antigen for target by the antibody bispecific that induces antigen.

T cells that must be activated in the tumor are those adjacent to the vasculature, since this is the region more accessible to cells and it is also where the antibody Bispecific will be more concentrated. Activated T cells should secrete IFN-? which induces antigens from Class II on the vasculature of the adjacent tumor.

The use of a bispecific antibody (Fab'-Fab ') that has a branch directed against a tumor antigen and the other branch directed against CD28 is preferred at present. This antibody will crosslink CD28 antigen on cells T in the tumor which, when combined with a second signal (provided, for example, by IL-1 which is commonly secreted by tumor cells (Burrows and collaborators, 1991; Ruco et al., 1990), has shown that activates T cells through a path independent of CA 2+ inhibible CsA (Hess et al., 1991; June and collaborators, 1987; Bjorndahl et al., 1989).

The preparation of antibodies against several cytokine activation molecules is also well known in the technique. For example, the preparation and use of antibodies monoclonal anti-CD14 and anti-CD28 that have the ability to induce cytokine production by leukocytes have now been described by several laboratories (reviewed in Schutt et al., 1988; Chen et al 1990, and June and collaborators, 1990, respectively). Moreover, the Preparation of monoclonal antibodies that stimulate release of cytokine leukocyte through other mechanisms and others activating antigens (Clark et al., 1986; Geppert and collaborators, 1990).

In still other embodiments, the inventors take into account an alternative approach to suppress the expression of class II molecules, and selectively obtain expression of Class II molecule at the tumor site. This approach, which avoids the use of both cyclosporin A and an activation antibody bispecific, take advantage of the fact that the expression of Class II molecules can be effectively inhibited by suppressing the IFN-γ production by T cells, by example, through the use of an anti-CD4 antibody (Street et al., 1989). Using this embodiment, the IFN-? production is inhibited by administering anti-CD4, resulting in general suppression of the expression of Class II. Class II is then induced only at the site of tumor location, for example, using tumor-specific T cells that are only activatable within the tumor.

\hbox{10 mg/kg}

de peso corporal. Después de que se suprime la expresión de Clase II, se preparará e introducirá en la corriente sanguínea unos clones de célula T que produce IFN-\gamma (por ejemplo, T_{h}l o linfocito T citotóxico, CTL) específico para un antígeno expresado en la superficie de las células tumorales. Estas células T se localizan en la masa del tumor, debido a su capacidad de reconocimiento de antígeno y, después de este reconocimiento, liberan entonces IFN-\gamma. De esta manera, se restringe de nuevo la liberación de citoquina al tumor, limitando de este modo la expresión de las moléculas de Clase II a la vasculatura del tumor.In this mode of treatment, a human animal or patient will generally be pretreated with a dose of anti-CD4 that is effective in suppressing IFN-γ production and thereby suppressing the expression of Class II molecules. It is taken into account that the effective doses are, for example, of the order of about 4 to about

 \ hbox {10 mg / kg} 

body weight After the expression of Class II is suppressed, some T-cell clones producing IFN-? (Eg, T h or cytotoxic T lymphocyte, CTL) specific for an expressed antigen will be prepared and introduced into the bloodstream on the surface of tumor cells. These T cells are located in the tumor mass, due to their ability to recognize antigen and, after this recognition, then release IFN-γ. In this way, the release of cytokine to the tumor is again restricted, thereby limiting the expression of Class II molecules to the tumor vasculature.

T-cell clones that produce IFN-γ can be obtained from peripheral blood (Mazzocchi et al. 1990), however, a preferred source is from within the tumor mass (Fox et al., 1990). The presently preferred element for preparing this T cell clone is to remove a part of a patient's tumor mass; isolate cells, using collagenase digestion, when necessary; enriching leukocytes that infiltrate the tumor using density gradient centrifugation, followed by suppression of other subsets of leukocytes by, for example, treatment with specific antibodies and complement; and then expand the tumor infiltration leukocytes in vitro to provide the clone that produces IFN-γ. This clone will necessarily be immunologically compatible with the patient, and therefore will be well tolerated by it.

It is proposed that benefits will be achieved individuals also selecting a T cell clone that produces IFN-? High from leukocytes expanded by determining the cytokine secretion pattern of each individual clone every 14 days. To this end, the remaining clones they will be mitogenically or antigenically stimulated during approximately 24 hours and the culture supernatants are they will try, for example, using a specific sandwich technique linked immunosorbent enzyme assay (Cherwinski and collaborators, 1989), to determine the presence of IL-2, IFN-?, IL-4, IL-5 and IL-10. Clones that secrete high levels of IL-2 and IFN-?, The pattern of characteristic cytokine secretion of clones T H1. I know confirm the specificity of the tumor using assays of proliferation.

In addition, it will be preferred to use as the antibody anti-CD4 an anti-CD4 Fab, due to which will be removed from the body within 24 hours after the injection and thus will not suppress T cell clones of Tumor recognition to be administered later. The preparation of T cell clones having specificity of the tumor is generally known in the art, as exemplified by the production and characterization of T cell clones from infiltration of solid melanoma tumors (Maeda and collaborators, 1991).

Using any of the suppression methods MHC Class II induction, benefits will likely result additional to the fact that antibodies Anti-Class II injected intravenously do not seem reach epithelial cells or monocytes / macrophages in normal organs other than the liver and spleen. Presumably this It is because the vascular endothelium in most organs normal is narrow, it is not open as it is in the liver and the spleen, and thus antibodies must spread through the basement membranes to reach the positive cells of Class II Also, any B cell removal that may result, for example, after crosslinking, it is unlikely that pose a significant problem, since these cells return to fill from negative Class II parents (Lowe and collaborators, 1986). Still the removal of B cells, as is presented in patients with B lymphoma, does not cause obvious damage (Vitetta and collaborators, 1991).

In short, although the compositions and Coagulant tumor antibodies of the present invention are excellently simple, and do not require induction of antigens for its operability, the combined use of a bispecific antibody that induces antigen with this invention. These antibodies will usually be administered before Bispecific coagulation ligands of this invention.

In general, the most `` immunogenic '' tumors would be the most convenient tumors for the MHC Class II approach involving, for example, the cross-linking of T cells in the tumor through the bispecific antibody anti-CD28 / antitumor, because it is more likely that these tumors are infiltrated by T cells, a prerequisite for this method to be effective. Examples of tumors immunogenic solids include renal carcinomas, melanomas, a minority of breast and colon cancers, as well as possibly cancers with pancreatic, gastric, liver, lung and glial These tumors are known as `` immunogenic '' because there is evidence that they get immune responses in the host and they has found that they are docile to cellular immunotherapy (Yamaue and collaborators, 1990). In the case of melanomas and cancers of the large intestine, the most preferred antibodies for use in these cases would be B72.3 (anti-TAG-72) and PRSC5 / PR4C2 (anti-Lewis antigen) or 9.2.27 (antigen anti-melanoma Mr high).

For most solid tumors of any origin, an anti-CD14 approach that employs a macrophage / monocyte intermediary would be more convenient. This is because most tumors are rich in macrophages. Examples of macrophage-rich tumors include most of the carcinomas of the breast, colon and lung. Examples of antibodies Preferred antitumor for use in these cases would be anti-HER-2, B72.3, SM-3, HMFG-2, and SWA11 (Smith and collaborators, 1989).

(c) Inductable coagulant markers

Coagulants, such as thrombin, Factor IX / IXa, Factor X / Xa, plasmin and metalloproteinases, such as interstitial collagenases, stromelysins and gelatinases, also They act to induce certain markers. In particular, E-selectin, P-selectin, PDGF e ICAM-1 are induced by thrombin (Sugama and collaborators, 1992; Shankar et al., 1994).

Therefore, for this induction a bispecific anticoagulant / antitumor antibody. The antibody is will locate in the tumor through its binding activity with the tumor. The bispecific will then concentrate the coagulant, by example, thrombin, in the tumor, resulting in the induction of E-selectin and P-selectin in vascular endothelial cells of the tumor (Sugama et al., 1991; Shankar et al., 1994).

Alternatively, Factor addressing of tissue truncated to tumor cells or endothelium will induce deposition of thrombin inside the tumor. As thrombin is deposited, it can induce E-selectin and P-selectin on endothelial cells Vascular tumors.

(d) Antibodies to endothelial cell markers vascular

A direct means to recognize an objective of vasculature associated with disease, whether induced in the environment natural or by artificial means, it is through the use of a antibody that has binding affinity for the receptor particular cell surface, molecule or antigen. These include antibodies directed against all components of the cell surface that is known to be present on, by example, vascular endothelial cells of the tumor, which are induced or overexpressed in response to factors derived from tumor, and those that are induced by the subsequent manipulation of the man's hand

Anti-vWF recognizes the antigen VIII R Ag and stain 100% of the types of tumors presented and stain 100% of the vessels in the tumor and have a strong staining pattern in normal glasses FB5 recognizes the endosialin antigen and stains the 50% of the tumor types presented and stained 10 to 30% of the vessels in the tumor and have a staining pattern in normal vessels in the lymphoid organs. TP3 recognizes the 80 kDa antigen osteosarcoma related to antigen protein and stains 50% of the tumor types presented and stained 10 to 30% of the vessels in the tumor and has a strong staining pattern in normal vessels over the small blood vessels. BC-1 recognizes the antigen isoforms of fibronectin and stains 60% of the types of presented tumor and stained 10 to 30% of the vessels in the tumor and not It presents staining pattern in normal vessels. TV-1 recognizes the fibronectin antigen and stains 100% of the types of presented tumor and stained 100% of the vessels in the tumor and presents a strong staining pattern in all normal vessels. LM 609 recognize the vitronectin receptor α_ {v} \ beta_ {e} and stain 85% of the tumor types presented and stained 70 to 80% of the vessels in the tumor and has a medium staining pattern in vessels normal. TEC-11 recognizes endoglin and stains 100% of the tumor types presented and stained 100% of the vessels in the tumor and has a weak staining pattern in most normal vessels TEC110 recognizes VEGF antigens and stains 100% of tumor types presented and stained 100% of the vessels in the tumor and has a weak staining pattern in most vessels normal.

In a comparative antibody study monoclonal anti-EC (MAbs) in human tumors it was found that TEC 110, TV-1 and TEC11 were positive in gastrointestinal, parotid, sinus, ovary tumors uterine, lung and Hodgkin tumors. While FB-5 had a slight staining in tumors gastrointestinal and lung and was negative in other tumors mentioned. TP-3 was positive in tumors gastrointestinal and less in parotid, ovarian and tumor types Hodgkins tumors. BC-1 was positive for gastrointestinal tumors, as well as for reproductive tumors and respiratory IM 609 was positive in gastrointestinal tumors, of ovaries, uterines, lungs and Hodgkin tumors as well as tumors of the reproductive and respiratory system.

Two other antibodies that can be used in this invention are those described by Rettig et al. (1992) and Wang et al (1993) that target antigens not related of unknown function expressed in the vasculature of human tumors, but not in most normal tissues.

The antibody described by Kim et al. (1993) can also be used in this invention, particularly since this antibody inhibited angiogenesis and suppressed tumor growth in vivo .

Antibodies that have not been shown previously that are specific to human tumors also can use. For example, Venkateswaran et al. (1992) described the production of monoclonal antibodies anti-FGF. Xu et al. (1992) developed and characterized a panel of 16 monoclonal antibodies and polyclonal domain-specific and isoforms against isoforms of FGF receptor (flg). Massoglia et al. (1987) also gave know monoclonal antibodies against the FGF receptor.

(e) Generation of antibodies to the vasculature of the disease

In addition to using a known antibody, such as described above and others known and published in the scientific literature, an antibody can also be generated novel using standard immunization procedures, as described in greater detail later in the present description. To generate an antibody against a vascular marker antigen known, associated with a disease, an animal would be immunized with an immunogenic composition that comprises the antigen. This can be a membrane preparation that includes or enriches for the antigen; a relatively purified form of the antigen, a isolated from cells or membranes; a highly purified form of antigen, as obtained by a variety of stages of purification using, for example, an original antigen extract or a recombinant form of the antigen obtained from a cell recombinant host

The present invention also discloses other methods to generate an antibody against an antigen present in vasculature endothelial cells associated with disease, These methods are convenient for use even when the identity Antigen biochemistry remain unknown. These methods are exemplify through the generation of an antibody against endothelial cells of the vasculature of the tumor. A first element to achieve antibody generation in this way use a Preparation of vascular endothelial cells obtained from the site of tumor location of an animal or human patient. An experimental animal is simply immunized with a preparation of these cells and the antibodies thus produced are collected. The most useful way of this method is when they are selected subsequently specific antibodies, as can be achieved using conventional hybridoma technology and selecting against cells vascular endothelial tumors.

A discovery of the above method is that it mimics the tumor vasculature phenomenon in vitro , and where cell purification is not necessary. Using this method, the endothelial cells are subjected to tumor-derived products, which could be obtained from conditional medium or from the tumor, in cell culture rather than in animal. This method generally involves stimulating endothelial cells with conditioned tumor medium and employing stimulated endothelial cells as immunogens to prepare a collection of antibodies. Again, specific antibodies must be selected, for example, using conventional monoclonal antibody technology, or other techniques such as combinatorial immunoglobulin phagemid libraries prepared from RNA isolated from the spleen of the immunized animal. A specific antibody that preferentially recognizes the stimulated vascular endothelium of the tumor and reacts more strongly with endothelial cells associated with the tumor than with normal adult human tissues would be selected.

(f) Antndoglin antibodies

One of the inventors has prepared and isolated antibodies that have relative specificity for endothelium vascular tumor The monoclonal antibody called the antibody tumor endothelial cell 4 and endothelial cell antibody of tumor 11 (TEC4 and TEC11) were obtained using the previous method (U.S. Patent Application with serial number 08 / 457.229 and 08 / 457.031, each incorporated in the present description as reference). The antigen recognized by TEC4 and TEC11 was determined Finally, it was the endoglin molecule. The epitopes on Endogline recognized by TEC4 and TEC11 are present on the cell surface of HUVE cells stimulated, and only minimally present (or immunologically accessible) on the surface of unstimulated cells. Monoclonal antibodies have been previously grown against endoglin. However, analyzing the reactivity with HUVEC or HUVEC cell surface determinants activated by TCM through FACS or indirect immunofluorescence shows that the epitopes recognized by TEC-4 and TEC11 are different from those of a previous antibody called 44G4 (Gougos and Letarte, 1988).

(g) Use of endothelial cell binding ligands vascular

They can also be used as a component target the biological ligands that are known to bind or interact with cell surface molecules endothelial, such as growth factor receptors.

The growth factors or ligands that are take into account, useful as objectives in this regard include VEGF / VPF, FGF, TGF?, Ligands that bind to a TIE, fibronectin isoforms associated with tumor, dispersion factor, hepatocyte growth factor (HGF), platelet factor 4 (PF4), PDGF and TIMP.

Particularly preferred objectives are VEGF / VPF, the FGF family of proteins and TGFβ. Abraham and collaborators (1986) cloned FGF, which is therefore available as a recombinant protein. How it is released by Ferrara et al. (1991), four species of VEGFs having 121, 165, 189, and 206 amino acids.

(h) Objective of linked ligands

Antibodies or specific target ligands they can also be directed to any component that links to the vascular endothelial cell surface at a site of disease, such as a tumor. These components are exemplified by ligands and tumor-derived antigens, such as factors of growth, which bind to cell surface receptors specific already present in endothelial cells, or to receptors that have been induced, or overexpressed, on these cells as response to the tumor environment. The objectives associated with the Tumor vasculature can also be called binding factors of endothelial cells derived from the tumor.

A level of specificity required to successfully target an objective of disease because local endothelial cells get will induce to express, or reveal, receptors that are not present, or are underexpressed or masked, in endothelial cells normal. With tumors, greater specificity will result due to fact that the endothelial cells in the tumor will capture the tumor-derived factors, and will link them to the surface cellular, reducing the amount of ligand available to others tissues. When combined with the additional dilution of the factor or ligand through distribution in blood and fluid culture of tissue, endothelial cells in normal tissues will be expected that link relatively few of these factors. In this way, Operationally, ligands or surface binding factors Cellular will be able to be used as cell markers endothelial tumors.

In addition to manufacturing by the same cells Tumor, tumor endothelial cell binding factors they can also originate from other types of cells, such as macrophages and mast cells, which have infiltrated tumors, or may be made by platelets that are activated within the tumor.

Other growth factors or ligands held for being useful as target substances associated with Tumor vasculature include EGF, FGF, VEGF, TGF?, HGF (NaKamura, 1991), angiotropin, TGF-?, TNF-?, PD-ECGF and ligands of TIE link (Bicknell and Harris, 1992). Target substances currently preferred are VEGF / VPF, and the FGF family of proteins, transforming the growth factor? (TGF-?); TGF-?; factor of tumor necrosis-? (TNF-?); angiotropin; growth factor of platelet-derived endothelial cell (PD-ECGF); TIE binding ligands; pleiotropin In addition, components of non-antibody target, such as annexins and peptides that comprise the tripeptide sequence R-G-D, which specifically direct to the vasculature of the tumor (Pasqualini et al., 1997), are also intended for use in certain aspects of the invention.

Another aspect of the present invention is the use of target antibodies, or binding regions thereof, that they are specific for epitopes present only in complexes ligand-receptor, these epitopes are absent both the individual (free) ligand and the receptor in its form not bound. These antibodies recognize and bind to the conformation only that results when a ligand, such as a factor of growth, binds to its receptor, such as a factor receptor of growth, to form a specifically bound complex. These epitopes are not present in uncomplexed forms of the ligands or receptors.

The inventors take into account that the complexes ligand-receptor to which these antibodies are link are present in a significantly higher number in endothelial cells associated with tumors than in cells Endothelial not associated with tumors. These antibodies so both will be useful as objective substances and will serve to increase additionally the specificity of bispecific coagulants of the invention.

(i) Receiving constructions

Soluble binding domains of endothelial cell surface receptors are also taken into account for use as target ligands in the present invention. This concept is generally based on the well-known sandwich bonding phenomenon that has been exploited in a variety of in vitro and in vivo binding protocols. Basically, since endothelial cells express specific receptors, the cells bind and adsorb the corresponding ligands, the ligands are then available to bind to other receptor constructs if they have to be introduced into the system.

A range of endothelial cell receptors useful has been identified in the previous sections, being particularly preferred objectives VEGF / VPF, FGF, TGFβ, TIE-1 and TIE-2. Each one of these receptors could be manipulated to form a link domain soluble for use as a target ligand.

iv. Stromal cell targets associated with the disease (a) Objectives of extracellular matrix / stroma

The usefulness of membrane markers basement in tumor pathology was described by Birembaut and collaborators (1985). These studies showed that the distribution of basement membrane (BM) markers, type IV collagen, laminin (LM), heparan proteoglycan sulfate (HSP) and fibronectin (FN) were interrupted in tumor pathology. Burtin and collaborators (1983) also described alterations of the basement membrane and connective tissue antigens in nodules human metastatic lymphatics.

A preferred target for use with the Invention is RIBS. Ugarova and collaborators (1993) unveiled conformation changes occur in fibrinogen and it produced by their interaction with membrane glycoprotein of platelet GPIIb-IIIa. The fibrinogen bond to GPIIb-IIIa glycoprotein membrane in platelets activated leads to platelet aggregation. This interaction gives as a result conformation changes in fibrinogen as evidence for the expression of binding sites induced by receptor, RIBS, epitopes that are expressed by the link but not for the free ligand.

Two RIBS epitopes have been located by Ugarova and collaborators (1993). A sequence resides in γ112-119 and is recognized by MAb 9F9; the second is the RGDF sequence in A? 95-98 and is recognized by mAb 155B16. These epitopes are also exposed by adsorption of fibrinogen on plastic surface and digestion of the molecule per plasmin. The proteolytic exposure of epitopes coincides with the dissociation of carboxyl terminal aspects of the A? chains to form the X2 fragment. The inaccessibility of the RGDF sequence of the A? sequence 95-98 in fibrinogen suggests that this sequence does not participates in the initial bond of the molecule to GPIIb-IIIa.

Fibrinogen binding to its receptor alters the conformation of the carboxyl terminal aspects of the chains A?, Exposing the sequences that reside in the segments spiral connectors between domains and D of the molecule, generating RIBS epitopes. In practical terms, the RIBS sequences as epitopes for use in directing them with a coaguligating Monoclonal antibodies 9F9 and 155B16 can be use this way advantageously, as well as antibodies described by Zamarron et al. (1991).

(b) Additional cellular objectives

The combinations for the use of this invention have the additional advantage that they can be used for direct coagulants to the vasculature associated with the disease directing them to cell types found within the region of the illness.

Platelets participate in hemostasis and in thrombosis adhering to the walls of blood vessels injured and accumulating at the site of the injury. Although the Platelet deposition at sites of blood vessel injury is responsible for the primary arrest of bleeding under conditions physiological, can lead to vascular occlusion with damage to resulting ischemic tissue and low thrombus embolization pathological conditions

Platelet interactions with your environment and each other represent complex processes that begin in the cell surface Therefore, the surface membrane provides a reactive interface between the external medium, which includes components of the blood vessel wall, and plasma and the inside of the platelet.

p-155, a platelet protein multimeric that is expressed in activated platelets (Hayward and collaborators 1991), can be attacked using the invention. The platelets respond to a large number of stimuli suffering complex biochemical and morphological changes. These changes are involve in physiological processes that include adhesion, aggregation, and coagulation. Platelet activation produces membrane alterations that can be recognized by monoclonal antibodies. Monoclonal antibody JS-1 (Hayward et al., 1991) is one of these antibodies intended for use as part of a coaguligand.

Ligand-induced binding sites (LIBS) they are sites expressed in cell surface receptors only after the ligand bond causes the receptor change shape, through subsequent biological events. These they can be seen as counterpart to the RIBS and are also objective preferred for use in the present invention.

Frelinger et al. (1990; 1991), developed 13 anti-LIBS antibodies, any of which can be used to administer a coagulant to a site of disease or tumor according to the present description. Murine monoclonal antiplatelet antibodies MA-TSPI-1 (directed against the human thrombospondine) and MA-PMI-2, MA-PMI-1, and MA-LIBS-1 (directed against LIBS in human platelet glycoprotein IIb / IIIa) from Dewerchin and collaborators (1991) can also be used, as well as RUU 2.41 and LIBS-1 by Heynen et al. (1994); OP-G2 by Tomiyama et al. (1992); Y Ab-15

Many other objectives, such as antigens or smooth muscle cells, pericytes, fibroblasts, macrophages and infiltration lymphocytes and leukocytes can also be used.

v. Toxins

For certain applications, the second therapeutic substances will be pharmacological substances bound to antibodies or growth factors, particularly cytotoxic or otherwise anti-cellular substances that have the ability to kill or suppress the growth of cell division of endothelial cells. In general, the secondary aspects of the invention take into account the use of any substance pharmacological that can be conjugated to a target substance, preferably an antibody, and actively administer to the endothelium or stroma target. Anticellular substances by title Examples include chemotherapeutic substances, radioisotopes as well. as cytotoxins In the case of chemotherapeutic substances, the inventors propose that substances such as a hormone, such like a steroid; an antimetabolite such as arabinoside cytosine, fluorouracil, methotrexate or aminopterin; an anthracycline, mitomycin C; a vinca alkaloid; demecolcina; etoposide; mitramycin; or an antitumor alkylating substance such as Chlorambucil or melphalan, will be particularly preferred. Other embodiments may include substances such as cytokine, factor of growth, bacterial endotoxin or the lipid fraction A of the bacterial endotoxin In any case, it is proposed that the substances such as these, if desired, can be successfully conjugated to an objective substance, preferably an antibody, in a manner that allows addressing, internalization, release or presentation to blood components at the site of target endothelial cells as required, using technology known conjugation (see, for example, Ghose et al., 1983 and Ghose et al 1987).

In certain preferred embodiments, the immunotoxins will generally include a plant-derived toxin, fungus or bacteria, such as chain A toxins, a ribosome inactivation protein, α-sarcin, aspergillin, restrictedtocin, a ribonuclease, diphtheria toxin or exotoxin pseudomonas , to mention only a few examples. The use of toxin antibody constructs is well known in the art of immunotoxins, as is its binding to antibodies. Of these, a particularly preferred toxin for binding with antibodies will be a deglycosylated ricin A chain. The deglycosylated ricin A chain is preferred because of its extreme potency, longer half-life, and because it is economically feasible to manufacture it in clinical and scale grade.

(a) Preparation of substance conjugates target-toxin

While the immunotoxin preparation is, in general, well known in the art (see, for example, Patents USES. 4,340,535, and European Patent number 44167, both incorporated herein by reference), the inventors are aware that certain advantages can be achieved through the application of certain preferred technology, both in the preparation of immunotoxins as in their purification for subsequent clinical administration. For example, although IgG-based immunotoxins will typically show better capacity of binding and slower elimination in the blood than its counterparts Fab ', immunotoxins based on Fab' fragments generally will exhibit better tissue penetration capacity compared with IgG based immunotoxins.

Additionally, although numerous types of linkers containing disulfide bond are known, which can be used successfully to conjugate the toxin fraction with the target substance, certain ligands will generally be preferred over other ligands, based on different pharmacological characteristics and capabilities. For example, ligands containing a disulfide bond that is sterically `` hindered '' will be preferred, due to their greater stability in vivo , thus preventing the release of the toxin fraction prior to binding at the site of action. Furthermore, although certain advantages according to the invention will be realized through the use of any of several toxin fractions, the inventors have found that the use of ricin A chain, and even more preferably the deglycosylated A chain, will provide benefits. private individuals

A wide variety of cytotoxic substances are known that can be conjugated with endothelial anti-cell antibodies. Examples include numerous toxins derived from plant, fungus, or even bacteria, which, by way of example, include several A-chain toxins, particularly ricin A chain, ribosome inactivation proteins such as saporin or gelonin, α-sarcin , aspergiline, restrictocin, ribonucleases such as placental ribonuclease, angiogenic, diphtheria toxin, and pseudomonas exotoxin, to name just a few. The most preferred toxin fraction for use in connection with the invention is the toxin A chain that has been treated to modify or eliminate carbohydrate residues, the so-called deglycosylated A chain. The inventors have had the best success through the use of deglycosylated ricin A chain (dgA) which is now commercially available from Inland Laboratories, Austin, TX.

However, it may be desirable from a point of pharmacological view employ the smallest possible molecule that without However provide an adequate biological response. It can wishing then to use smaller chain A peptides the which will provide an adequate anti-cellular response. With this Finally, it has been discovered by others that ricin A chain can be `` truncate '' by removing 30 amino acids from terminal N by nagarase (Sigma), and still retain a toxin activity adequate. It is proposed that when desired, this truncated A string is It can be used in conjugates according to the invention.

Alternatively, you can find that the application of recombinant DNA technology to the chain fraction Toxin A will provide additional significant benefits of according to the invention. Since the cloning and expression of the Biologically active ricin A chain has been enabled through from the publication of others (O'Hare et al., 1987; Lamb and collaborators, 1985; Halling et al., 1985), is now possible to identify and prepare smaller peptides or otherwise variants that however exhibit adequate toxin activity. Moreover, the fact that ricin A chain has now been cloned allows the application of site-directed mutagenesis, through which can be easily prepared and selected for peptides chain A derivatives and obtain additional useful fractions for its use in relation to the present invention.

Crosslinking of the A chain region of conjugate toxin with the region of the target substance is a important aspect of the invention. In certain cases, it is required that a crosslinker that has a disulfide function be used by the conjugate to have biological activity. The reason for this is not clear, but probably due to a need for certain toxin fractions are easily releasable from the substance objective once the substance has `` administered '' the toxin to target cells Each type of regulator, as well as the way in that the crosslinking is performed, the pharmacodynamics will tend to vary of the resulting conjugate. Finally, in cases where you have considering a releasable toxin, it is desired to have a conjugate that remain intact under conditions found everywhere in the body except at the intended site of action, at this point it is desirable that the conjugate have good characteristics of `` liberation. '' Therefore, the particular crosslinking scheme, including in particular the particular crosslinking reagent used and the structures that are crosslinked, will have some importance.

Depending on the specific toxin compound used as part of the fusion protein, it may be necessary provide a peptide separator operably linked to the target substance and the toxin compound that is capable of bend into a disulfide bonded fork structure. The proteolytic dissociation inside the fork will then produce a heterodimeric polypeptide wherein the target substance and the toxin compound are bound by only a single disulfide bond (See for example, Lord et al., 1992). An example of this toxin is the ricin A chain toxin.

When other toxin compounds are used, a non-dissociable peptide separator can be provided to operatively bind the target substance and the toxin compound of the fusion protein. Toxins that can be used together with non-dissociable peptide spacers are those that can themselves be converted by proteolytic dissociation into a form linked by cytotoxic disulfide (see, for example, Ogata et al., 1990). An example of a toxin compound is an exotoxin compound of Pseudonomas .

Nucleic acids can be used in the present description comprising nucleic acid sequences encoding an objective substance of interest and sequences of nucleic acid encoding a toxin substance of interest. These nucleic acid sequences encoding target substance and that encode toxin substance bind in a way that the translation of the nucleic acid produces the substance compounds target / toxin of the invention.

(b) Union of other substances to substances objective

It is taken into account that most of the therapeutic applications of immunotoxin aspects Additional aspects of the present invention will involve the direction of a fraction of toxin to the endothelium of the tumor or stroma. This is due to the largest capacity of most toxins in administer a cell killing effect compared to others potential substances However, there may be circumstances, such as when the target antigen is not internalized by a route consistent with efficient poisoning by target substance / toxin compounds, such as immunotoxins, where you will want to direct substances chemotherapeutics such as antitumor drugs, other cytokines, antimetabolites, alkylating substances, hormones, and the like. The advantages of these substances over their conjugated counterparts of non-objective substances is the added selectivity that the target substance, such as an antibody. You could mention example title substances such as steroids, cytosine arabinoside, methotrexate, aminopterin, anthracyclines, mitomycin C, vinca alkaloids, demecolcina, etoposide, mitramycin, and the like. The list, of course, is only by way of example because the technology to link pharmaceutical substances to target substances, such as antibodies, for specific administration to tissues it is well established (see, for example, Ghose and Blair, 1987).

A variety of chemotherapeutic substances and other pharmacological substances have now successfully conjugated to antibodies and shown to work pharmacologically (see, for example, Vaickus et al 1991). The substances Antineoplastic examples that have been investigated include dexorubicin, daunomycin, methotrexate, vinblastine, and several others (Dillman et al., 1988; Pietersz et al., 1988). Plus yet, the binding of other substances such as neocarzinostatin (Kimura et al., 1983), macromycin (Manabe et al., 1984), trenimón (Ghose, 1982) and α-amanitin (Davis and Preston, 1981).

saw. Coaguligands

The second target substance for optional use with the invention it can also comprise an objective component that Be able to promote coagulation. These `` substances that promote objective coagulation '' or `` coaguligands '' include any of the above target substances that are Operationally associated with one or more coagulation factors. The target substance may be directly linked to a factor that Directly or indirectly stimulate coagulation, or substance target can be linked to a second link region that is able to bind and release a coagulation factor that directly or indirectly stimulate coagulation.

(a) Coagulation factors

Coagulation factors as an example are the types of truncated, dimeric tissue factor molecules, multimeric and mutant of the present invention, as described in The present description in detail.

A variety of other factors of coagulation in relation to the present invention, as exemplified by the substances indicated below. When a coagulation factor covalently binds to a first binding substance or target, a site other than its Functional coagulation site to bind the molecules. The regions suitable binding other than active sites, or regions Functional, coagulation factors are also described in each of the following sections.

Coagulation factors

In the present invention they can also be used thrombin, Factor V / Va and derivatives, Factor VIII / VIIIa and derivatives, Factor IX / IXa and derivatives, Factor X / Xa and derivatives, Factor XI / XIa and derivatives, Factor XII / XIIa and derivatives, Factor XIII / XIIIa and derivatives, activator of Factor X and activator of Factor V.

Venom coagulants

Williams and Esnouf, in 1962, demonstrated that the Russell's viper venom contains a clotting protein. Kisiel (1979) isolated a venom glycoprotein that activates Factor V; Y Di Scipio et al. (1977) showed that a protease of Venom activates the human X Factor. The activator of Factor X is the component intended for use in this invention.

Monoclonal antibodies specific to the Factor X activator present in Russell's viper venom have also occurred (for example, MP1 of Pukrittayakamee and collaborators, 1983), and could be used to administer the substance at a specific target site within the body.

Prostaglandins and synthetic enzymes

Thromboxane A2 is formed of endoperoxides through the sequential actions of cyclooxygenase enzymes and thromboxane synthetase in platelet microsomes. Thromboxane A_ {2} is easily generated by platelets and is a potent vasoconstrictor, by virtue of its ability to produce aggregation of platelets (Whittle et al., 1981).

Both thromboxane A2 and the like assets thereof are intended for use herein invention. A synthetic protocol to generate thromboxane A2 is described by Bhagwat et al. (1985). Analogs of thromboxane A2 described by Ohuchida et al. (1981) (especially compound 2) are particularly intended for Use with this description.

It is possible that thromboxane synthase, and others enzymes that synthesize prostaglandins that activate platelets, They can also be used as `` coagulants '' in the present context. Shen and Tai (1986) describe monoclonal antibodies, and the immunoaffinity purification of thromboxane synthase; and Wang and collaborators (1991) discloses the cDNA for thromboxane synthase human

Fibrinolysis inhibitors

Α2-antiplasmin, or α2-plasmin inhibitor, is an inhibitor of Proteinase naturally present in human plasma that works to efficiently inhibit lysis of fibrin clots induced by the plasminogen activator (Moroi and Aoki, 1976). The α2-antiplasmin is an inhibitor particularly powerful, and is intended for use herein invention.

The α2-antiplasmin can be purified as first described by Moroi and Aoki (1976). Other purification schemes are also available, such as using plasminogen-sepharose affinity chromatography, ion exchange chromatography on DEAE-sefadex and concanavalin-A-sepharose chromatography; or using affinity chromatography on a sepharose column having an elastase digested plasminogen formulation that contains three triple N- terminal structures in the plasmin A chain (LBSI), followed by gel filtration (Wiman and Collen, 1977 ; Wiman, 1980, respectively).

As the cDNA sequence of the α2-antiplasmin is available (Tone and collaborators, 1977), a preferred method for the production of α2-antiplasmin will be through expression recombinant

Antibodies are also available monoclonal against α2-antiplasmin which can use in the embodiments of bispecific binding ligand of the invention. For example, Hattey et al. (1987) describe two monoclonal antibodies against α2-antiplasmin, MPW2AP and MPW3AP. Since each one of these monoclonal antibodies was disclosed that they react equally well with the original α2-antiplasmin, both could be used to administer α2-antiplasmin exogenous to a target site or accumulate endogenous α2-antiplasmin and concentrate it within the target region. Other ones could also be used antibodies such as JTPI-2, described by Mimuro and collaborators

(b) Substances that bind coagulation factors

Another group of objective coagulation ligands For use with the Fabric Factors of this invention are those in which the target region is not directly linked to a coagulation factor, but is linked to a second binding region that binds to a coagulation factor.

When a second link region is used for bind and administer a coagulation factor, the binding region is choose so that it recognizes a site in the coagulation factor that does not significantly incapacitate its ability to induce coagulation. The regions of coagulation factors convenient to link in this way will generally be the same as regions that are convenient for the link covalent to the target region, as described in the sections previous.

However, since the bispecific ligands of this class can be expected to release the clotting factor after administration to the site or region of the tumor, there are more allowable flexibility in the clotting factor regions suitable for binding to a second substance or antibody of link.

The second link regions suitable for its use in this way will generally be combination sites of Antigen of antibodies that have binding specificity for the coagulation factor, including the functional parts of the antibodies, such as scFv, Fv, Fab ', Fab and fragments F (ab ') 2.

The bispecific binding ligands that contain antibodies, or fragments thereof, directed against Tissue Factor, thrombin, precaliquein, V / Va Factor, Factor VIII / VIIIa, Factor IX / IXa, Factor X / Xa, Factor XI / XIa, Factor XII / XIIa, Factor XIII / XIIIa, Russell's viper venom, the thromboxane A2 or α2-antiplasmin are exemplary embodiments of this aspect of the invention.

(c) Tissue Factor Prodrugs

\hbox{(Y) _{n2} }

Z ligando, en donde tTF_{1-219} representa el Factor de Tejido menos los dominios citosólico y de transmembrana; X representa un dominio de transmembrana hidrofóbica con n1 aminoácidos (AA) de tamaño (n=1-20 AA); Y representa una secuencia de reconocimiento de proteasa hidrofílica de n2 aminoácidos de tamaño (suficientes aminoácidos para asegurar el reconocimiento adecuado de proteasa); Z representa un tioéster de disulfuro u otro grupo de enlace tal como (Cys)_{1-2}; Ligando representa un anticuerpo u otra fracción objetivo de reconocimiento de células tumorales, tumor EC, tejido conectivo (estroma) o marcadores de lámina basal.Examples of truncated Tissue Factor prodrugs have the following structures: tTF_ {1-219} (X) n1

 \ hbox {(Y) n2} 

Z ligand, wherein tTF 1-219 represents the Tissue Factor minus the cytosolic and transmembrane domains; X represents a hydrophobic transmembrane domain with n1 amino acids (AA) in size (n = 1-20 AA); And represents a hydrophilic protease recognition sequence of n2 amino acids in size (sufficient amino acids to ensure adequate protease recognition); Z represents a thioester of disulfide or other linking group such as (Cys) 1-2; Ligand represents an antibody or other target tumor cell recognition fraction, EC tumor, connective tissue (stroma) or basal lamina markers.

The truncated Tissue Factor prodrug is account for intravenous injection, allowing Locate the diseased tissue (for example, tumor). As soon as locates in the diseased tissue, endogenous proteases (for example, metalloproteinases, thrombin, Factor Xa, Factor VIIa, Factor IXa, plasmin) will dissociate the protease recognition sequence hydrophilic prodrug which will allow the sequence of Hydrophobic transmembrane is inserted into a local cell membrane. Once the tail has been inserted into the membrane, the Factor of Truncated tissue will recover its induction properties of coagulation, resulting in clot formation in the Vasculature of the desired tissue.

(d) Bispecific Antibodies

In general, antibody preparation Bispecific is also well known in the art, as exemplify Glennie et al. (1987). Antibodies bispecific have been used clinically, for example, to treat cancer patients (Bauer et al., 1991). A method for bispecific antibody preparation involves preparation separate from antibodies that have specificity for the antigen of target tumor cell, on the one hand, and the clotting substance (or another desired target, such as an activating antigen) by the other.

Bispecific antibodies have also been developed particularly for use as substances immunotherapeutic As mentioned above in conjunction with antigen induction, certain of these antibodies are developed to reticulate lymphocytes and tumor antigens (Nelson, 1991; Segal et al., 1992). Examples include chimeric molecules that bind T cells, for example, in CD3, and tumor antigens, and trigger lymphocyte activation by the physical cross-linking of the TCR / CD3 complex in close proximity with the target cell (Staerz et al., 1985; Perez and collaborators, 1985; 1986a; 1986b; Ting et al., 1988).

Undoubtedly, tumor cell carcinomas, lymphomas, leukemias and melanomas have been known to be susceptible to bispecific antibody mediated elimination by T cells (Nelson, 1991; Segal et al., 1992, deLeij et al., 1991). These types of antibodies Bispecifics have also been used in several clinical trials of phase I against various tumors targets. Antibodies Bispecific crosslinking can be administered as described in references such as deLeij et al. (1991); Clark and collaborators (1991); Rivoltini et al. (1992); Bolhuis and collaborators (1992); and Nitta et al. (1990).

Although numerous methods are known in the art for the preparation of bispecific antibodies, the method of Glennie et al. (1987) involves the preparation of F (ab 'γ) 2 peptide fragments of the two antibodies chosen, followed by the reduction of each one to provide fragments of Fab '\ gamma_ {SH} separately. The SH groups in one of the two partners to mate are rented with a crosslinking reagent such as o-phenylenedimeleimide to provide groups Free maleimide in a partner. This partner can be conjugated with the other through a thioether bond, to give the desired heteroconjugate F (ab 'γ) 2.

Due to the ease of preparation, high performance and reproducibility, the method of Glennie et al. (1987) is often preferred for antibody preparation bispecific, however, there are numerous different approaches that are They can employ and they are considered by the inventors. By For example, other techniques are known where the cross-linking with SPDP or protein A, or a construct is prepared triespecifica (Titus et al. 1987; Tutt et al., 1991).

Another method to produce antibodies Bispecific is by merging two hybridomas to form a quadroma (Flavell et al., 1991, 1992; Pimm et al., 1992; French et al., 1991; Embleton et al., 1991). As used in the present description, the term `` quadroma '' is used to describe the productive fusion of two hybridomas of cells B. Using now standard techniques, two hybridomas are fused together produce antibody to produce daughter cells, and are selected those cells that have maintained the expression of both sets of clonotype immunoglobulin genes.

A preferred method to generate a quadroma involves the selection of an enzyme deficient mutant of how At least one of the parent hybridomas. This first cell line of mutant hybridoma is then fused with cells of a second hybridoma that has been lethally exposed, for example, to iodoacetamide, preventing its continued survival. The fusion cell allows the rescue of the first hybridoma acquiring the gene for its enzyme deficiency from the treated hybridoma lethally, and the rescue of the second hybridoma through fusion with the first hybridoma. Fusion is preferred, but not required of immunoglobulins of the same isotype, but of a subclass different. A mixed subclass antibody allows use if there is an alternative test for the isolation of a quadroma preferential.

In greater detail, a method of developing quadroma and selection involves obtaining a hybridoma line that secretes the first monoclonal antibody chosen and makes it deficient for the essential metabolic enzyme, hypoxanthine-guanine phosphoribosyltransferase (HGPRT). To obtain hybridoma deficient mutants, they grow cells in the presence of increasing concentrations of 8-azaguanine (1x10-7 M at 1 x 10-5 M). The mutants are subcloned by limiting dilution and testing their sensitivity to hypoxanthine / aminopterin / thymidine (HAT). The culture medium may consist of, for example, DMEM supplemented with 10% fetal calf serum (FCS), 2 mM L glutamine and 1mM of penicillin-streptomycin.

A complementary hybridoma cell line which produces the second desired monoclonal antibody is used to generate the quadromas by cell fusion techniques standards (Galfre et al., 1981), or using the protocol described by Clark et al. (1988). In short, they mix 4.5 x 10 7 first hypoxanthine sensitive cells / aminopterin / thymidine with 2.8 x 10 7 second cells resistant to hypoxanthine / aminopterin / thymidine that have been previously treated with a lethal dose of the biochemical inhibitor irreversible iodoacetamide (5 mM in regulated saline solution of phosphate) for 30 minutes on ice before melting. The fusion cell is induced using polyethylene glycol (PEG) and cells are plate in 96-well microculture plates. The quadromas are select using medium containing hypoxanthine / aminopterin / thymidine Cultures containing antibody are identified bispecific using, for example, an isotype specific ELISA of solid and stained phase of specific immunofluorescence for isotype

In an embodiment of identification for identify bispecific antibody, plate wells of microtiter (Falcon, Becton Dickinson Labware) are coated with a reagent that interacts specifically with one of the parent hybridoma antibodies and lacking cross reactivity With both antibodies. The plates are washed, blocked, and the Supernatants (SNs) to be tested are added to each well. The plates are incubated at room temperature for two hours, the supernatants are discarded, the plates are washed, and diluted phosphatase conjugate alkaline-anti-antibody added for 2 hours at room temperature. The plates are washed and add a phosphatase substrate, for example, P-Nitrophenylphosphate (Sigma, St. Louis) at each well. The plates are incubated, 3N NaOH is added to each well to stop the reaction, and OD410 values are determined using an immunosorbent linked enzyme assay reader (ELISA).

In another embodiment of identification, they are used microtiter plates pretreated with poly-L-lysine to join one of the target cells to each well, then the cells are fixed, by example using 1% glutaraldehyde and antibodies tested bispecific to determine its ability to bind the cell intact In addition, FACS, staining by immunofluorescence, idiotype specific antibodies, antigen binding competition, and other common methods in the antibody characterization technique, together with the present invention to identify the preferred squares.

After the isolation of the quadroma, it purify bispecific antibodies away from other products cell phones. This can be accomplished through a variety of protein isolation procedures, known for experts in the art of immunoglobulin purification. The means for preparing and characterizing antibodies are well known in the technique (see, for example, Antibodies: A Laboraty Manual, 1988).

For example, the supernatants of quadromas selected are passed on columns of sepharose protein A or G protein to bind IgG (depending on the isotype). Then the antibodies are eluted with, for example a citrate regulator with pH 5.0. Eluted fractions containing antibodies Bispecific dialysate against an isotonic regulator. Alternatively, the eluate is also passed over a column of sepharose anti-immunoglobulin. Antibodies Bispecific eluted with 3.5 M magnesium chloride. Bispecific antibodies purified in this way are tested for determine its binding activity by, for example, an assay Isotype specific bound enzyme immunosorbent and assay of immunofluorescence staining of the target cells, as described above

The purified bispecific antibodies and the parent antibodies can also be characterized and isolated by SDS-PAGE electrophoresis, followed by staining with Silver or Coomassie. This is possible when one of the antibodies parents have a molecular weight greater than the other, where the band of bispecific antibodies migrates midway between that of The two parent antibodies. Sample reduction verifies the presence of heavy chains with two different molecular weights apparent.

In addition, technology is now available recombinant for the preparation of antibodies in general, allowing the preparation of recombinant antibody genes that encode an antibody that has the desired dual specificity (Van Duk et al., 1989). Thus, after selecting the monoclonal antibodies that have the binding characteristics more preferred, the respective genes can be isolated for these antibodies, for example, by immunological selection of a phage expression library (Oi and Morrison, 1986; Winter and Milstein, 1991). Then, through the redisposition of the domains Fab coding, you can easily get the construction adequate chimeric.

vii. Combined treatment

Tissue Factor compositions in combination with either immunotoxins or coaguligands are intended for use in the clinical treatment of several cancers humans and even other disorders, such as prostatic hyperplasia benign and rheumatoid arthritis, in which term detention Medium or longer blood flow would be advantageous.

The combination of factor compositions tissue described in the present application with immunotoxins and coaguligands are considered to be tools particularly useful in antitumor therapy. From the data presented in this description, including animal studies, and the knowledge in the art regarding lymphoma treatment (Glennie et al., 1988), can be developed directly appropriate doses and treatment regimens aiming T cells (Nolan and Kennedy, 1990) and aiming drugs (Paulus, 1985).

It is currently proposed that the effective doses of immunotoxins and coaguligands for use with the constructions of tissue factor described above in the treatment of cancer will be between about 0.1 mg / kg and about 2 mg / kg, and preferably, between about 0.8 mg / kg and approximately 1.2 mg / kg, when administered through the route IV at a frequency of about once a week. Will happen necessarily some variation in dosage depending on the state of the subject being treated. The person responsible for the administration, in any case, will determine the appropriate dose for the individual subject. This optimization and adjustment leads to performed routinely in the technique and in no way reflects An undue amount of experimentation.

Naturally, before its use extends extensively, animal studies and trials will be carried out Additional clinics The different elements to drive a clinical trial, including patient treatment and supervision, will be known to those skilled in the art to the Light of the present description. The following information is being presenting as a general guideline for use in the establishment of these essays.

It is taken into account that the patients chosen for combined studies they would have failed to respond to at least a conventional therapy course and they must have disease Objectively measurable as determined by physical examination, techniques laboratory, or radiographic procedures. When they are used parts of murine monoclonal antibody in immunotoxins or coaguligands, patients should not have had a history of Mouse immunoglobulin allergy. Any chemotherapy should stop at least two weeks before entering the study.

With regard to the administration of tissue factor constructs, either with immunotoxins or coaguligands, it is considered that certain advantages will be found in the use of a resident central venous catheter with a gate of triple lumen. The therapeutic mixtures should be filtered, by example, using a 0.22 µ filter, and diluted properly, such as with saline solution, up to a final volume of 100 ml. Before use, the test sample should also be filtered from in a similar way, and its concentration be assessed before and after the filtration determining the A 280. The expected recovery It should be within the range of 87 to 99%, and adjustments will be considered for protein loss.

These combinations of tissue factor and immunotoxin or coaguligand can be administered for a period approximately 4-24 hours, receiving each patient of 2 to 4 infusions in intervals of 2 to 7 days. The administration can also be carried out through a regime stable infusion over a period of 7 days. The infusion given to any dose level should depend on any toxicity observed. Therefore, if grade II toxicity is reached after any simple infusion, or over a period of time particular for an infusion of stable regime, should be retained other doses or stop infusion at a steady regimen until the toxicity improve. Increasing doses of tissue factor either with immunotoxins or coaguligands should be administered to groups of patients until approximately 60% of patients show unacceptable toxicity grade III or IV in any category. The doses that are 2/3 of this value should be defined as the dose safe.

Physical exam, tumor measurements, and tests laboratory, of course should be done before treatment and at intervals of up to a month later. Laboratory tests should include complete blood counts, serum creatinine, creatine kinase, electrolytes, urea, nitrogen, SGOT, bilirubin, albumin, and total whey protein. The serum samples taken up to 60 days after treatment should be evaluated by radioimmunoassay to determine the presence of tissue factor intact, immunotoxin and / or coaguligating or components thereof and antibodies against any part thereof. Analysis immunological serum, using any standard assay such as, for example, an immunosorbent enzyme linked assay or radioimmunoassay, will allow to evaluate the drug-kinetics and substance elimination therapy.

To evaluate antitumor responses, you have Please note that patients should be examined within 48 hours at one week and again 30 days after the last infusion. When a palpable disease is present, should be measured daily two perpendicular diameters of all the masses during the treatment, within a week after completing therapy, and after 30 days To measure non-palpable disease, they will be performed CT scans in series at intervals of one centimeter across the chest, abdomen, and pelvis at 48 hours up to a week and again at 30 days. Tissue samples should also be evaluated. histologically, and / or by flow cytometry, using biopsies of disease sites or even blood or fluid samples If it is suitable.

Clinical responses can be defined by acceptable measurement. For example, a complete answer can be define by the disappearance of every measurable tumor one month after treatment While a partial response can be define by a reduction of 50% or greater of the sum of the products of perpendicular diameters of all modules Tumor evaluable one month after treatment, without sites of tumor location showing enlargement. By way of similar, a mixed response can be defined by a reduction in product of perpendicular diameters of all measurable lesions for 50% or more one month after treatment, with progress in one or more sites

F. Prolonged half-life of the Tissue Factor

It is shown in the present description that the antitumor activity of the truncated tissue factor is increased combining the truncated tissue factor with carrier molecules, such as immunoglobulins, which delay the elimination of the factor of truncated body tissue. For example, linking the factor of truncated immunoglobulin tissue increases antitumor activity prolonging the live half-life of the truncated tissue factor of so that the truncated tissue factor persists for longer and has more time to induce thrombotic events in the vessels Tumor The prolongation in the half-life is the result of either increase in size of the truncated tissue factor above the threshold for glomerular filtration; or the active re-absorption of conjugated within the kidney, a property of the Fc piece of immunoglobulin (Spiegelberg and Weigle, 1965). It is also possible that the immunoglobulin component change the conformation of the factor of truncated tissue to make it more active or stable. They are held in counts other carrier molecules in addition to immunoglobulin to produce similar effects and are then included within the present invention

F1 Modifications

Since a first interpretation of life prolonged mean observed after tissue factor binding truncated to immunoglobulin is simply that the increase resulting in size leads to prolonged half-life of plasma, the inventors take into account that other modifications that increase the size of the tissue factor constructs can be used of advantageously in relation to the present invention, as long as the elongation modification do not substantially restore the membrane binding functionality with the construction of factor modified tissue In the absence of such a possibility, that can easily be tested, virtually any molecule generally biologically acceptable inert can be conjugated with a Fabric Factor construction in order to prepare a Modified tissue factor with increased live half-life.

The modification can also be made to the Structure of the Fabric Factor itself to make it more stable, or maybe to reduce the catabolism regime in the body. A mechanism for these modifications is the use of d-amino acids instead of l-amino acids in the Tissue Factor molecule. Technicians with ordinary experience in the field will understand that the Introduction of these modifications needs to be followed by rigorous testing of the resulting molecule to ensure that It still retains the desired biological properties. Other stabilizing modifications include the use of the addition of stabilizing fractions either at the N-terminal or at C-terminal or both, which is generally used to prolong the half-life of biological molecules. A way For example only, you may want to change the terms of the Fabric Factor constructs by acylation or amination. The variety of these modifications can also be used together, and parts of the molecule can also be replaced Tissue Factor by peptidomimetic chemical structures that give as a result the maintenance of biological function and still Improve the stability of the molecule.

F2 Conjugates i. Protein

Useful techniques in relation to proteins of conjugation of interest for carrier proteins are used widely in the scientific community. It will generally be understood that in the preparation of these tissue factor conjugates for the use in the present invention, the protein chosen as a carrier molecule must have certain defined properties. By example, of course it must be biologically compatible and not give as a result no significant opposite effect after the administration to a patient In addition, it is generally required that the carrier protein is relatively inert, and not immunogenic, both properties will result in the maintenance of the Function of the Tissue Factor and will allow the construction resulting avoid excretion through the kidney. Proteins to Sample titles are albumins and globulins.

ii. No protein

In common with the protein conjugates described above, the Tissue Factor molecules of the present invention may also be conjugated to non-protein elements in order to improve their half-life in vivo . Again, the choice of non-protein molecules for use in these conjugates will readily be apparent to those skilled in the art. For example, one or more of a variety of natural or synthetic polymers, including polysaccharides and polyethylene glycol, can be used.

In the context of preparing conjugates, either Protein or non-protein, care should be taken that the introduced conjugate does not substantially reassociate the molecule from Tissue factor modified with the plasma membrane so that increase your coagulation capacity and result in a molecule that exerts harmful side effects after administration. As a general rule, it is believed that hydrophobic additions or conjugates largely should be avoided in relation to these realizations

iii. Immunoconjugates

When antibodies are used to conjugate the Truncated Tissue Factor compositions of the present invention, the choice of antibody will generally depend on the intended use. of the tissue factor-antibody conjugate. By example, when immunoconjugates of Tissue Factor are destined to its use in addition to the tissue factor molecules alone, the type Tumor should be considered, for example, if it is preferable target the tumor cells, or more preferably, the tumor vasculature or tumor stroma. As immunoconjugates of the Tissue Factor are the therapeutic substances themselves primary, immunoconjugates will in no way be a `` target immunoconjugate. '' In these aspects, the conjugation of the Tissue Factor molecule to an antibody or part thereof It is simply carried out in order to generate a construction that has improved half-life and / or bioavailability in comparison with the original Tissue Factor molecule. In any case, it they can achieve certain advantages through the application of types particular antibodies. For example, although it is expected that IgG-based antibodies exhibit better binding abilities and removal in the blood slower than its Fab 'counterparts, the compositions based on Fab 'fragments generally exhibit better tissue penetration capacity.

(a) Monoclonal antibodies

The elements to prepare and characterize antibodies are well known in the art (See, for example, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, 1988).

The methods to generate monoclonal antibodies (MAbs) generally begin along the same lines as those of preparing polyclonal antibodies. In short, an antibody polyclonal is prepared by immunizing an animal with a composition immunogenic according to the present invention, with or without previous immunotolerance, depending on the antigen composition and of the protocol being used (for example, tolerating a population of normal cells and then immunize them with a population of tumor cells), and collect the antiserum from the animal immunized A wide range of animal species can be used to the production of antiserum. Typically the animal used for antiserum production is a rabbit, a mouse, a rat, a Hamster, a guinea pig or a goat. Due to volume relatively large blood of rabbits, a rabbit is a preferred choice for antibody production polyclonal

As is well known in the art, a composition given may vary in its immunogeneicity. Often is therefore it is necessary to raise the host's immune system, as can be achieved by coupling a peptide or immunogen polypeptide to a carrier. Preferred and exemplary carriers are the keyhole limpet hemocyanin (KLH) and serum albumin bovine (ASB). Other albumins can also be used as carriers such as egg albumin, mouse serum albumin or albumin of rabbit serum. The elements to conjugate a polypeptide to a carrier protein are well known in the art and include glutaraldehyde, ester of m-maleimidobenzoyl-N-hydroxysuccinimide, carbodiimide and bis-biazotized benzidine.

As is well known in the art, the immunogeneicity of a particular immunogenic composition can be increased by the use of non-specific immune response stimulators, known as adjuvants. Exemplary and preferred adjuvants include Freund's adjuvant (a non-specific stimulator of the immune response containing dead Mycobacterium tuberculosis ), incomplete Freund's adjuvants, and aluminum hydroxide adjuvant.

The amount of immunogen composition used in polyclonal antibody production varies by nature of the immunogen as well as of the animal used for immunization. I know you can use a variety of routes to administer the immunogen (subcutaneous, intramuscular, intradermal, intravenous and intraperitoneal). The production of polyclonal antibodies can be monitor by sampling blood of the immunized animal at various points after immunization. You can also give a second booster shot. The process of reinforcement and title is repeated until a convenient degree is achieved. When you get a desired titration level, the immunized animal can bleed and the serum is isolated and stored, and / or the animal can be used to generate monoclonal antibodies.

Antibodies can easily be prepared monoclonal by using well known techniques, such as those exemplified in U.S. Pat. No. 4,196,265. Typically, this technique involves immunization of an animal. suitable with a selected immunogen composition, by example, a purified or partially purified tumor cell, or vascular endothelial cell protein, polypeptide, peptide, or intact cell composition. The immunizing composition is administered in an effective way to stimulate cells that They produce antibodies. Rodents such as mice and Rats are the preferred animals, however, it is also possible the use of rabbit cells, sheep, frog. The use of rats can provide certain advantages (Goding, 1986, pages 60-61), but mice are preferred, being more preferred mice the BALB / c, since this one is used more routinely and generally gives a higher percentage of mergers stable.

After immunization, they are selected somatic cells with the potential to produce antibodies, specifically B lymphocytes (B cells), for use in the monoclonal antibody generation protocol. These cells can be obtained from spleens, tonsils or lymph nodes of biopsies, or from a peripheral blood sample. I know prefer spleen cells and peripheral blood cells, the first because they are a rich source of cells that produce antibodies that are in the stage of division of plasmablast, and the latter because peripheral blood is easily accessible. Frequently, a set of animals and the spleen of the animal with the titration of higher antibodies will be removed and lymphocytes will be obtained of the spleen homogenizing the spleen with a syringe. Typically, the spleen of an immunized mouse contains approximately 5 X 10 7 up to 2 X 10 8 lymphocytes.

B lymphocytes that produce antibodies to from the immunized animal they then fuse with cells of a immortal myeloma cell, usually one of the same species that The animal that was immunized. Myeloma cell lines suitable for use in fusion procedures that it produces hybridoma are preferably non-antibody producers, which they have high fusion efficiency, and enzyme deficiencies that become unable to grow in a certain selective medium that supports the growth of only the desired fused cells (hybridomas).

Any of several cells of myeloma, as those skilled in the art know (Goding, pages 65-66, 1986; Campbell, pages 75-83, 1984). For example, when the animal Immunized is a mouse, P3-X63 / Ag8 can be used, X63-Ag8.653, NS1 / 1.Ag 4 1, Sp210-Ag14, FO, NSO / U, MPC-11, MPC11-X45-GTG 1.7 and S194 / 5XX0 Bul; for rats R210.RCY3, Y3-Ag 1.2.3 can be used, IR983F, 4B210 or any of the mouse cell lines mentioned previously; and U-266, GM1500-GRG2, LICR-LON-HMy2 and UC729-6, are useful in relation to mergers human cell phones

The methods to generate spleen hybrids producer of antibody or lymph node cells and cells Myeloma usually involves mixing somatic cells with cells of myeloma in a 4: 1 ratio, although the ratio may vary from about 20: 1 to about 1: 1, respectively, in the presence of a substance or substances (chemical or electrical) that promote the fusion of membranes cell phones. The fusion methods that use Sendai virus have been described by Kohler and Milstein (1975; 1976), and those who use polyethylene glycol (PEG), such as 37% (volume / volume) of polyethylene glycol, by Gefter et al., (1977). The use of electrically induced fusion methods is also suitable (Goding pages 71-74, 1986).

Fusion processes usually produce viable hybrids at low frequencies, approximately 1 X 10-6 to 1 X 10-8. However, this does not pose a problem, since the fused, viable hybrids differ from non-molten, parent cells (particularly non-molten myeloma cells that would normally continue to divide indefinitely) by culturing them in a selective medium. The selective medium is generally one that contains a substance that blocks de novo synthesis of nucleotides in the tissue culture medium. Exemplary and preferred substances are aminopterin, methotrexate, and azaserine. Aminopterin and methotrexate block de novo synthesis from both purines and pyrimidines, while azaserine blocks only purine synthesis. When aminopterin or methotrexate is used, the medium is supplemented with hypoxanthine and thymidine as a source of nucleotides (HAT medium (`` hypoxanthine / aminopterin / thymidine '')). When azaserine is used, the medium is supplemented with hypoxanthine.

The preferred selection medium is hypoxanthine / aminopterin / thymidine. Only cells capable of operating these nucleotide restoration pathways are able to survive in the middle hypoxanthine / aminopterin / thymidine. The cells of Myeloma are defective in key enzymes of the restoration path, for example, hypoxanthine phosphoribosyl transferase (HPRT), and not They can survive. B cells can operate this route, but they have a limited life span in cultivation and generally They die within about two weeks. Therefore, the only cells that can survive in the selective medium are those hybrids formed of myeloma and B cells.

This crop provides a population of hybridomas from which specific hybridomas are selected. Typically, hybridoma selection is carried out by culturing cells by diluting a single clone in plates microtiter, followed by tests of clonal supernatants individual (after about two to three weeks) to Determine the desired reactivity. The essay could be sensitive, simple and fast, such as radioimmunoassays, immunoassays of enzyme, cytotoxicity assays, plaque assays, punctual immunolink, and the like.

Selected hybridomas would be serially diluted and cloned into cell lines that produce individual antibodies, whose clones can then propagate indefinitely to provide monoclonal antibodies. Cell lines can be exploited for the production of monoclonal antibodies in two basic ways. A hybridoma sample can be injected (frequently into the peritoneal cavity) into a histocompatible animal of the type that was used to provide the somatic and myeloma cells for the original fusion. The injected animal develops tumors that secrete the specific monoclonal antibody produced by the fused cell hybrid. Animal body fluids, such as serum or ascites fluid, can be drained to provide monoclonal antibodies in high concentration. Individual cell lines could also be cultured in vitro , where monoclonal antibodies are naturally secreted in the culture medium from which they can be easily obtained in high concentrations. Monoclonal antibodies produced by any means can be further purified, if desired, using filtration, centrifugation and various chromatographic methods such as HPLC (`` high pressure liquid chromatography '') or affinity chromatography.

The inventors also take into account the use of a molecular cloning approach to generate monoclonal. For this, immunoglobulin phagemid libraries are prepared combinatorial RNA isolated from the spleen of an immunized animal, and it select the phagemids that express the appropriate antibodies by flushing using cells that express the antigen and control cells for example, normal cells against tumor. The advantages of this approach over hybridoma techniques Conventional are that they can be produced and select 10 4 times more antibodies in a single round, and that new specificities are generated by the chain combination H and L which also increases the chance of finding antibodies adequate.

When monoclonal antibodies are used in the present invention, can be of human, murine, monkey, of rat, hamster, chicken or even rabbit. The invention provides the use of human antibodies, `` humanized '' antibodies or mouse, rat or other species chimerics, which have regions constant domain and / or human variable, and other antibodies recombinants and fragments thereof. Of course, due to the ease of preparation and immediate availability of reagents, monoclonal antibodies are typically preferred murine.

(b) Functional antibody binding regions Fab

Antibody fragments can be obtained Functional through complete immunoglobulin proteolysis by nonspecific thiolprotease, papain. Papain first must be activated by reducing the sulfydryl group at the active site with cysteine, 2-mercaptoethanol or dithiothreitol. Should be remove heavy metals in the enzyme material by chelation with EDTA (2 mM) to ensure enzymatic activity maximum The enzyme and the substrate normally mix with each other at a ratio of 1: 100 by weight. After incubation, the reaction can be stopped by irreversible alkylation of thiol group with iodoacetamide or simply by dialysis. Shall digestion termination monitored by SDS-PAGE and the different fractions separated by protein A-sepharose or chromatography of ion exchange

F (ab ') 2

The usual procedure for the preparation of F (ab ') 2 fragments of rabbit immunoglobulin G and of human origin is the proteolysis limited by the enzyme pepsin (Protocol 7.3.2.). The conditions, 100x excess antibody weight / weight in acetate regulator at pH 4.5, at 37 ° C, suggest that the antibody dissociates on the C-terminal side of the disulfide bond of heavy inter-chain The digestion rates of Mouse immunoglobulin G may vary with subclasses and may be difficult to obtain high yields of fragments F (ab ') 2 active without some undigested immunoglobulin G or completely degraded. In particular, IgG_b is very susceptible to complete degradation. The other subclasses require different incubation conditions to produce results optimal.

Digestion of rat IgG by pepsin requires conditions that include dialysis in 0.1 M regulator acetate, pH 4.5, and then incubation for 4 hours with 1% of weight / weight of pepsin; the digestion of IgG1 and IgG2a improves if it is first dialyzed with respect to the 0.1 M format regulator, pH 2.8, a 4 ° C, for 16 hours followed by acetate regulator. IgG_b gives more consistent results with V8 protease incubation of Staphylococcus (3% weight / weight) in 0.1 M sodium phosphate regulator, pH 7.8, for 4 hours at 37 ° C.

iv. Second generation of Factor immunoconjugates Tissue

The inventors take into account that the Fc part of the immunoglobulin in the construction of Truncated Tissue Factor-immunoglobulin employed in the advantageous studies described in the present description can actually be the relevant part of the antibody molecule, resulting in a half-life. Live increased. It is reasonable to presume that the conjugation of the Fc region results in active readsorption of a TF-Fc conjugate into the kidney, restoring the conjugate to systemic circulation. As such, any of the deficient coagulation tissue factor constructs or variants of the invention can be conjugated to an Fc region in order to increase the in vivo half-life of the resulting conjugate.

Several methods are available to produce Fc regions of sufficient purity to allow conjugation at Fabric Factor constructions. As an example only, chemical dissociation of antibodies to provide domains defined or parts are well known and easily practiced, and recombinant technology can also be used to prepare substantial amounts of Fc regions or, no doubt, prepare the whole TF-Fc conjugate after generation of a recombinant vector that expresses the fusion protein desired.

Other manipulations of the structure of General immunoglobulin can also be carried out with the in order to provide the second generation of constructions of Tissue Factor with increased half-life. As an example only, you can consider replacing the domain C_ {H} 3 of a IgG molecule with a Truncated Tissue Factor or a variant of the same. In general, the most effective mechanism to produce this hybrid molecule will use molecular cloning techniques and recombinant expression All these techniques are generally known to those skilled in the art, and are described in detail in the present description.

F3 Liaison means

The above compositions can be linked to Tissue Factor compositions in any operative manner that allows each region to perform its intended function without significant deterioration of the functions of the Tissue Factor. From In this way, the link components will be able to prolong the half-life of the construction, and the Fabric Factor is capable of Promote blood clotting.

i. Biochemical crosslinkers

The union of any of the components above, to a composition of Tissue Factor generally will use the same technology as the one developed for the preparation of immunotoxins. It is considered as a general guideline that any biochemical crosslinker that is suitable for use in a immunotoxin will also be useful in the present context, and also may consider additional linkers.

Crosslinking reagents are used to form molecular bridges that tie together functional groups of two different molecules, for example, a stabilizing substance and coagulant. To bind two different proteins in a way staggered, crosslinkers can be used hetero-bifunctional that eliminate the formation of unwanted homopolymers.

TABLE IV Crosslinkers hetero-functional

       \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {| l | l | l | c |} \ hline
 Linker \ + Reagent Towards \ + Advantages and Applications \ +
Distance of the \\ \ + \ + \ + separating arm \\ \ + \ + \ +
then \\ \ + \ + \ + cross-linking \\\ hline SMPT \ + Amines
Primary \ + x Greater stability \ + 11.2 A \\ \ + sulfydryls \ +
\ + \\\ hline SPDP \ + Primary amines \ + x Thiolation \ + 6.8 A \\
 \ + sulfydryls \ + x Crosslinking \ + \\ \ + \ + dissociable \ +
\\\ hline LC-SPDP \ + Primary amines \ + x
Extended separation \ + 15.6 A \\ \ + sulfydryls \ + \ + \\\ hline
Sulfo-LC- \ + Primary amines \ + x Separation
extended \ + 15.6 A \\ SPDP \ + sulfydryls \ + x Water soluble
\ + \\\ hline SMCC \ + Primary amines \ + x Reactive group \ + 11.6
A \\ \ + sulfydryls \ + stable maleimide \ + \\ \ + \ + x
Enzyme conjugation- \ + \\ \ + \ + Antibody \ + \\ \ + \ + x
Conjugation of Hapten- \ + \\ \ + \ + carrier protein \ + \\\ hline
 Sulfo-SMCC \ + Primary amines \ + x Group
reagent \ + 11.6 A \\ \ + sulfydryls \ + stable maleimide \ + \\
 \ + \ + x Water soluble \ + \\ \ + \ + x Enzyme conjugation- \ + \\
  \ + \ + Antibody \ + \\\ hline MBS \ + Primary amines \ + x
Enzyme conjugation- \ + 9.9 A \\ \ + sulfydryls \ + Antibody \ +
\\ \ + \ + x Conjugation Hapten- \ + \\ \ + \ + carrier protein
\ + \\\ hline Sulfo-MBS \ + Primary amines \ + x
Soluble in water \ + 9.9 A \\ \ + sulfydryls \ + \ + \\\ hline SIAB
\ + Primary amines \ + x Enzyme conjugation- \ + 10.6 A \\ \ +
sulfydryls \ + antibody \ + \\\ hline Sulfo-SIAB
\ + Primary amines \ + x Water soluble \ + 10.6 A \\ \ +
sulfydryls \ + \ + \\\ hline SMPB \ + Primary amines \ + x
Extended separation \ + 14.5 A \\ \ + sulfydryls \ + x
Enzyme conjugation- \ + \\ \ + \ + antibody \ + \\\ hline
Sulfo-SMPB \ + Primary amines \ + x Separation
extended \ + 14.5 A \\ \ + sulfydryls \ + x Water soluble \ +
\\\ hline EDC / Sulfo- \ + Primary amines \ + x Conjugation Hapten-
\ + 0 \\ NHS \ + Carboxyl groups \ + carrier \ + \\\ hline ABH \ +
Carbohydrates no \ + x Reacts with groups \ + 11.9 A \\ \ +
selective \ + sugar \ +
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
    

An example of crosslinker hetero-bifunctional contains two reactive groups: one that reacts with the primary amine group (for example N-hydroxysuccinimide) and the other that reacts with a thiol group (for example, pyridyl disulfide, maleimides, halogens, etc.). Through the primary amine reactive group, the crosslinker can react with the lysine residues of a protein (for example, the selected antibody or fragment) and through the thiol reactive group, the crosslinker, already bound to the first protein, reacts with the cysteine residue (free sulfhydryl group) of the another protein (for example, the coagulant).

Therefore you can see that the composition Preferred Fabric Factor will generally have, or will be derived to have, a functional group available for crosslinking purposes. This requirement is not considered to be limiting, since a wide Variety of groups can be used in this way. For example, groups primary or secondary amine, hydrazide or hydrazine groups, alcohol carboxylic, phosphate, or alkylating groups can be used to bonding or crosslinking. For an overview of the technology of link, you may wish to refer to Ghose and Blair (1987).

The separating arm between the two reactive groups of a crosslinker can have different sizes and compositions chemical A longer separating arm allows better flexibility of conjugate components while some particular components in the bridge (for example, benzene group) they can lend extra stability to the reactive group or a resistance increased chemical bond to action of various aspects (by example, disulfide bond resistant to reducing substances). The use of peptide separators, such as how L-Leu-L-Ala-L-Leu-L-Ala.

It is preferable to use a crosslinker that has reasonable stability in the blood. Numerous types of disulfide bonds containing linkers that can be used as Successful way to combine target substances and coagulants. The linkers containing a disulfide bond that is prevented sterically they can try to give greater stability live, avoiding Tissue Factor release before joining the site of action. These linkers are then a preferred group of binding substances.

One of the most crosslinking reagents Preferred for use in immunotoxins is SMPT, which is a bifunctional crosslinker that contains a disulfide bond that is `` sterically hindered '' by an adjacent benzene ring and methyl groups It is believed that steric linkage impairment disulfide serves a link protection function of the attack of thiolate anions such as glutathione, which may be present in tissues and blood, and by means of which it helps avoid decoupling of the conjugate before administration of the substance bound to the tumor location site. It has note that the SMPT substance can also be used in relation to with the bispecific coagulant ligands of this invention.

The SMPT crosslinking reagent, along with others Many known crosslinking reagents, lends the ability to cross-link functional groups such as cysteine SH or primary amines (for example, the amino epsilon group of lysine). Another possible type of crosslinker includes phenylazides hetero-bifunctional photoreactive containing a dissociable disulfide bond such as sulfosuccinimidyl-2- (p-azido salicylamide) ethyl-1,3'-dithiopropionate. The group N-hydroxy-succinimidyl reacts with the primary amino groups and phenylazide (after photolysis) reacts nonselectively with any residue of amino acid

In addition to the crosslinkers prevented, it also may use non-impeded linkers in accordance with this description. Other useful crosslinkers, not considered for contain or generate a protected disulfide, include SATA, SPDP and 2-iminothiolane (Wawrzynczak and Thorpe, 1987). The use of these crosslinkers is very well understood in the art.

As soon as it is conjugated, the Tissue Factor truncated is usually purified to separate the conjugate from non-conjugated or coagulant or other objective substances pollutants A large number of purification techniques are available for use to provide one-degree conjugates Enough of purity to make them clinically useful. Methods purification based on size separation, such as gel filtration, gel permeation or liquid chromatography High performance, will generally be of much use. I also know they can use other chromatographic techniques, such as separation by Blue-Sepharose.

ii. Recombinant Fusion Proteins

The truncated Tissue Factor compositions of the invention can also be fusion proteins prepared by molecular biology techniques. The use of recombinant DNA techniques to achieve these ends is now a common practice for those skilled in the art. These methods include, for example, in vitro recombinant DNA techniques, synthetic techniques and live recombination / genetic recombination. Additionally, DNA and RNA synthesis can be performed using an automatic synthesizer (see, for example, the techniques described in Sambrook et al., 1989; and Ausubel et al., 1989).

The preparation of this fusion protein generally involves the preparation of a first and a second DNA coding regions and functional ligation or binding of said regions, in frame, to prepare a single region of encoding encoding the desired fusion protein. In the present context, the Truncated Tissue Factor or the sequence of Mutant DNA of Tissue Factor will generally be boxed together with the DNA sequence encoding an inert protein carrier, immunoglobulin, Fc region, or something similar. Generally not believed that is particularly relevant if the Fabric Factor part of the fusion protein or the inert part is prepared as the region N-terminal or as the region C-terminal In relation to the second generation of Tissue Factor immunoglobulin molecules, the sequences of Fabric Factor coding may also be inserted within the immunoglobulin coding regions, so that the tissue factor sequences functionally interrupt the immunoglobulin sequences and the encoded protein can be Consider a `` Tribute. ''

As soon as the coding of the desired region, an expression vector is created. The vectors of expression contain one or more promoters upstream of the regions of inserted DNA that act to promote transcription of DNA and thus promote protein expression recombinant encoded. This is the meaning of `` expression recombinant '' and has been discussed elsewhere in the specification.

F4 essays

As with other aspects of the present invention, as soon as a candidate tissue factor construction has been generated with the intention of providing a living construction increased live media, construction should generally be tested to ensure that the desired properties have been imparted to the resulting compound. The different essays for your use to determine these changes depending on routine are and the Those skilled in the art easily put them into practice.

In the tissue factor conjugates designed simply in order to increase its size, confirmation of your Increased size is completely routine. For example, simply the candidate composition will be separated using any methodology which is designed to separate biological components based on the size and separate products will be analyzed in order to determine that a Fabric Factor construction has been generated of increased size. By way of example only, you can mention separation gel and separation columns, such as gel filtration columns. The use of filtration columns in gel in the separation of mixtures of conjugated components and not conjugates may also be useful in other aspects of this invention, such as in the generation of levels relatively high conjugates, immunotoxins or coaguligands.

Since the objective of the present class of conjugates is to provide a deficient coagulation Tissue Factor molecule that has an increased live half-life, the modified variants of the candidate Tissue Factor or the conjugates should generally be tested in order to confirm that it is Present this property. Again, these tests are routine in the art. A first test would be to determine the half-life of the modified tissue factor or candidate conjugate in an in vitro test. These assays generally comprise mixing the candidate molecule in serum and determining whether or not the molecule persists in a relatively intact form for a longer period of time, compared to the initial sample of Deficient Coagulation Tissue Factor. Again, sample aliquots of the mixture will be sampled and their size and, preferably, their biological function will be determined.

Live trials of biological half-life or `` removal '' can easily be carried out. In these systems, it is generally preferred to label the constructions of Tissue factor test candidates with a detectable marker and follow the presence of the marker after administration at animal, preferably through the intended route in the final therapeutic treatment strategy. As part of this process, body fluid samples would be taken, particularly serum and / or urine samples, and the samples would be analyzed for determine the presence of the marker associated with the construction of the Tissue Factor, which would indicate the longevity of the construction in the natural environment in the body.

One or more of a combination of molecules of the Fabric Factor with increased half-life can be used in this way together with the therapeutic methods described herein description. The proposed doses for administration they will generally be between about 0.2 milligrams and approximately 200 milligrams per patient, as with Original Fabric Factor constructions described previously. However, since these Factor molecules Tissue have been modified, it is possible that effective doses may be still smaller, such as about 0.1 milligrams Treatment regimens are more likely Therapeutics are altered when using Life Tissue Factors increased mean in the number of times the drugs are administered, instead of an alteration of the given dose. For example, when an original Fabric Factor construction is proposed for its use on days 1, 3 and 7 within the treatment period, the Improved counterpart tissue factor with longer half-life is would administer only on day 1 and on day 7. On any case, all these optimizations in terms of dose and timing of administration will be easily determined by technicians with field experience

G. Combinations of Tissue Factor and Factor VIIa

The inventors have further demonstrated that the activity that induces coagulation of truncated Tissue Factor bound to A20 cells markedly increased in the presence of Factor VIIa. In common with previous studies, these in vitro results are also transferred to the live environment. The studies are presented in the present description to demonstrate that the antitumor activity of several deficient coagulation tissue factor constructs increases after co-administration with Factor VIIa. Even using an experimental animal model of the HT29 tumor, which is remarkably difficult to coagulate, co-administration of the deficient coagulation Tissue Factor and exogenous Factor VIIa constructs resulted in considerable necrosis of the tumor tissue.

These data can be explained since the Factor of truncated tissue joins Factor VII but does not mediate efficiently its activation to Factor VIIa by Xa and the molecules of Adjacent factor VIIa. Providing a source of Factor VIIa preformed (exogenous) this block is overcome, allowing a more efficient coagulation. The success of the combined treatment of Deficient Coagulation Tissue Factor and Factor VIIa it is usually based on the amazing location of the Tissue Factor construction within the tumor vasculature. In the absence of this surprising location and functional effects specific, the co-administration of Factor VIIa would not have meaning in the context of tumor treatment, and may still be harmful as it promotes unwanted thrombosis in various tissues healthy. The combined use of the Truncated Tissue Factor and the Factor VIIa in an unaddressed manner has been previously proposed in relationship with the treatment of hemophiliacs and patients with others blood disorders, in which there is a fundamental deterioration of The coagulation cascade. In the present invention, the cascade of coagulation is usually fully operational, and the therapeutic invention concentrates this activity within a region defined body.

Therefore another object is to increase the effects antitumor of any of the tissue factor constructs of the invention combining the use of the Fabric Factor with the additional administration of Factor VIIa. Since the Fabric Factor truncated joins the vascular endothelium of the tumor, it is possible to inject Truncated Tissue Factor in animals that have a tumor, wait for a period of time for excess tissue factor to be removed truncated, and then inject Factor VIIa to magnify the action Trombotic Tissue Factor truncated into the vessels of the tumor. In this way, the Truncated Fabric Factor or other Construction of poor coagulation tissue factor can be see that it forms a reserve within the tumor, which allows the subsequent administration of Factor VIIa to increase and perpetuate the antitumor effect.

Another observation of the present invention is that the thrombotic activity of the Truncated Tissue Factor Factor VII (G164A) and Truncated Tissue Factor (W158R) activation mutants was largely restored by Factor VIIa. These mutations are within a region of the truncated Tissue Factor that is important for the conversion of Factor VII to Factor VIIa. As with the same Truncated Tissue Factor, studies in the present description show that the addition of preformed Factor VIIa often overcomes this blockage in the formation of coagulation complex. The present description exploits this and the aforementioned observations with the aim of publicizing in vivo cancer therapy.

Undoubtedly, the studies presented in the present description confirm that co-administration of a mutant variant of Factor VII activation of Tissue Factor with preformed Factor VIIa results in considerable necrotic damage to tumors, even in small tumor models that they are not the most docile for treatment with the present invention. This aspect of the invention is particularly surprising, since it was not previously believed that these mutants would have no therapeutic utility in any other embodiment than, perhaps, in the competitive inhibition of Tissue Factor as can be used to inhibit or reduce coagulation. Apart from this hypothesis, the generation of these mutants has been motivated by scientific interest and could perhaps be used as controls in certain in vitro studies. Only studies of the present inventors make these mutants clinically useful, either in the context of targeted administration (WO 96/01653), or in the even more surprising combined uses of the present invention.

In particular embodiments, this application, therefore, it involves first injecting tTF (G164A), tTF (W158R) or an equivalent thereof in animals that have tumors Then the tissue factor mutant is allowed Truncated is located in the tumor vessels and the residue is removed. Then, this is followed by the injection of Factor VIIa, which allows the truncated Tissue Factor mutants to be located express thrombotic activity. This strategy offers the advantage of That is very safe. Truncated Tissue Factor Mutants They are practically non-toxic, just like Factor VIIa. In this way, administration of the truncated Tissue Factor mutant followed Factor VIIa will be harmless to the host, although it efficiently induces thrombosis in the tumor vessels.

G1. Factor VIIa

Factor VII can be prepared as described Fair (1983), and as shown in U.S. Pat. numbers 5,374,617, 5,504,064 and 5,504,067. The coding part of the Human Factor VII cDNA sequence was released by Hagen and collaborators, (1986). The amino acid sequence from 1 to 60 corresponds to the pre-pro / forward sequence that is removed by the cell before secretion. The mature chain of Factor VII polypeptides consist of amino acids 61 to 466. The Factor VII becomes its active form, Factor VIIa, by dissociation of a single peptide bond between arginine-212 and isoleucine-213.

Factor VII can be converted in vitro to Factor VIIa by incubating the purified protein with Factor Xa immobilized in Affi-Gel MR 15 beads (Bio-Rad). The conversion can be monitored by SDS-polyacrylamide gel electrophoresis of small samples. Free Factor Xa in the preparation of Factor VIIa can be detected with the chromogenic substrate methoxycarbonyl-D-cyclohexyl glycyl-glycyl-arginine-p-nitroanilide (Spectrozyme MR Factor Xa, American Diagnostica, Greenwich, CT) at a final concentration of 0.2 mM in the presence of 50 mM EDTA.

Recombinant Factor VIIa can also be buy at Novo Biolabs (Danbury, CT).

G2 Treatment

The use of Factor VIIa in relation to The present invention is not confined to its ability to improve significantly the usefulness of the activation mutants of Factor VII described in the present description. It also has note that Factor VIIa will be used together with the molecules of Coagulation tissue factor deficient in equivalent activity to the Truncated Tissue Factor used first. In these treatment embodiments, the dose of Factor construction Tissue will generally be between approximately 0.2 milligrams and approximately 200 milligrams per patient. Adequate doses for Factor VIIa they can be determined better in light of this information.

For example, you may want to create a proportion 1: 1 of the construction of Tissue Factor and Factor VIIa in a precomplex and administer the precomplexed composition to the animal. If this is desired, an amount of Factor of Tissue and a sufficient amount of Factor VIIa to allow formation of an equimolar complex. To achieve this, it must be preferably using a 2-3 molar excess of Factor VIIa in order to ensure that each of the Factor molecules Tissue fits properly. Then it would simply separate Uncomplexed Tissue Factor and Mix Factor VIIa complexed using any convenient technique, such as gel filtration After the formation of the TF complex: VIIa, the complex can simply be administered to a patient who need treatment at a dose of approximately 0.2 milligrams and approximately 200 milligrams per patient.

As indicated above, generally it may be preferred to administer in advance a construction of A poor coagulation tissue factor to a patient, leaving the enough time to tissue factor to locate specifically within the tumor. After this pre-administration, then an adequate dose of Factor VIIa sufficient to coordinate and complex with the Tissue Factor located inside of the vasculature of the tumor. Again, the dose of Factor VIIa in order to allow a 1: 1 molar ratio of Tissue Factor and Factor VIIa is formed in the tumor environment. Given the differences in molecular weight of these two molecules, it will be seen that it would be advisable to add approximately twice as many amount in milligrams of Factor VIIa compared to milligrams of the tissue factor.

However, the above analysis is only a example title, and any dose of Factor VIIa that you give as result generally an improvement in coagulation It would obviously be of clinical importance. Regarding this, it is notable that the studies presented in the present description of made use a 16: 1 tissue factor surplus compared to Factor VIIa, which is generally approximately a surplus 32-fold molar of the tissue factor construction. Without However, notable coagulation and necrosis were observed specifically in the tumor Therefore, it will be clear that the effective doses of Factor VIIa are quite broad. As an example only, you may consider giving a patient a dose of Factor VIIa between about 0.01 milligrams and about 500 milligrams per patient

Each of the above analyzes can be also apply to the use of tissue factor constructions that have been mutated to impair their ability to activate the Factor VII. Since the above calculations are based on a proportion of link, it is not considered necessary to use particularly increased levels of Factor VIIa in combination with activation mutants described. However, since the administration of Factor VIIa It is not believed to be particularly harmful in itself, the potential to use increased doses of Factor VIIa is certainly evident.

Although the detailed guide released previously believed to be enough to allow a person with ordinary experience in the technique know how to put in practice these aspects of the invention, it can also be done reference to other quantitative analyzes to help in the Optimization of the doses of the Tissue Factor and Factor VIIa for administration. By way of example only, U.S. Pat. numbers 5,374,617; 5,504,064; and 5,504,067 describe a range of Therapeutically active doses and plasma levels of Factor VIIa.

Morrissey and Comp have announced that, in the context of bleeding disorders, the Tissue Factor of poor coagulation can be administered in an effective dose to produce an effective level of between 100 nanograms / milliliter and 50 micrograms / milliliter, or one level preferred between 1 microgram / milliliter and 10 micrograms / milliliter or 60 to 600 micrograms / kilogram weight body, when administered systemically; or an effective level between 10 micrograms / milliliter and 50 micrograms / milliliter or a preferred level between 10 micrograms / milliliter and 50 micrograms / milliliter, when administered topically (Patent USES. No. 5,504,064).

Factor VIIa is administered in a dose effective to produce an effective level of between 20 nanograms / milliliter and 10 micrograms / milliliter, (1.2 to 600 micrograms / kilogram), or a preferred level of between 40 nanograms / milliliter and 700 micrograms / milliliter (2.4 to 240 micrograms / kilogram) or a level between 1 microgram of Factor VIIa / milliliter and 10 micrograms of Factor VIIa / milliliter when administered topically.

In general, Tissue Factor of poor coagulation and activator of Factor VII to produce levels of up to 10 micrograms of Coagulation Tissue Factor deficient / milliliter of plasma and between 40 nanograms and 700 micrograms of Factor VIIa / milliliter of plasma. Although these studies were conducted in the context of bleeding disorders, they also have relevance in the context of the present invention, since the levels must be effective but supervised adequately to avoid systemic toxicity due to elevated levels of poor coagulation tissue factor and Factor VIIa activated. Therefore, the Factor VII activator is administered at an effective dose to produce a level in plasma Factor VIIa cash, as defined above.

G3 Factor VII activators

As described in U.S. Pat. number 5,504,064, endogenous Factor VII activators can also be administer instead of the same Factor VIIa. As described in the Previous patent, Factor VIIa can also be formed live, shortly before, at the time of, or preferably shortly after of the administration of Coagulation Tissue Factors deficient. In these embodiments, the endogenous Factor VII is converts to Factor VIIa by infusing an activator of the Factor VIIa, such as Factor Xa (FXa) in combination with phospholipids (PCPS `` phosphatidyl choline / phosphatidylserine '').

Factor VII activators in vivo include Factor Xa / PCPS, Factor IXa / PCPS, thrombin, Factor XIIa and the Factor VII activator for Oxyuranus scutellatus venom} in combination with phosphatidylcholine / phosphatidylserine. These have been shown to activate Factor VII in Factor VIIa in vitro . The activation of Factor VII to Factor VIIa for Xa / PCPS in vivo has also been measured directly. In general, the Factor VII activator is administered in a dose between 1 and 10 micrograms / milliliter of carrier (US Patent No. 5,504,064).

Phospholipid can be provided in several forms such as phosphatidylcholine / phosphatidylserine vesicles (PCPS) Phosphatidylcholine vesicle preparations / phosphatidylserine and the method of administration of Xa / PCPS is described in Giles et al., (1988), whose ideas are incorporated specifically in the present description. Other preparations of phospholipids can be replaced by PCPS, as long as they accelerate the Factor VII activation by Factor Xa. The effectiveness, and so Both determining the optimal composition and dose, you can monitor as described below.

A very effective dose of Xa / PCPS, which raises the Factor VIIa levels live in the chimpanzee, has been given to know that it is 26 pmoles FXa + 40 pmoles phosphatidylcholine / Serine phosphatidyl per kilogram of body weight. That dose produced an 18-fold increase in endogenous Factor VIIa levels (up to 146 nanograms / milliliter). An effect was observed marginally detectable using a smaller dose in dogs, where the infusion of 12 pmoles of Factor Xa + 19 pmoles of phosphatidylcholine / phosphatidylserine per kilogram of body weight produced a threefold increase in Factor VIIa levels endogenous. In accordance with the above, the doses of Factor Xa that they are at least 12 pmoles of Factor Xa per kilogram of weight body, and preferably 26 pmoles of Factor Xa per kilogram of body weight should be useful. The doses of phosphatidylcholine / phosphatidylserine which are at least 19 pmoles of phosphatidyl choline / phosphatidylserine per kilogram of body weight, and preferably 40 pmoles phosphatidylcholine / phosphatidylserine per kilogram of body weight, are equally useful (U.S. Patent No. 5,504,064).

You can monitor the effectiveness of any Factor VII activator in infusion after administration intravenous, taking samples of citrated blood at times variables (at 2, 5, 10, 20, 30, 60, 90 and 120 minutes) after a bolus infusion of the activator, and prepare plasma with few platelets for blood samples. The amount of Factor VIIa Endogenous can then be measured in citrated plasma samples performing a Factor VIIa coagulation test based on Factor of poor coagulation tissue. The desired Factor levels Endogenous VIIa would be the same as the target Factor levels VIIa in plasma indicated for coinfusion of purified Factor VII and The Clotting Factor of Poor Coagulation. Therefore, they could try other Factor VII activators live for generation of Factor VIIa with proper experimentation and dose adjustment to generate the desired Factor VIIa levels using the test of Coagulation Factor Factor deficient-based on Factor VIIa of plasma samples. The proper dose of Factor VII activator (which produces the desired Factor level Endogenous VIIa) can then be used in combination with Recommended amounts of Coagulation Tissue Factor deficient.

Doses can be timed to provide prolonged elevation in Factor VIIa levels. The dose preferably they would be administered until the effect is achieved desired antitumor, and then repeated as necessary to control bleeding The half-life of Factor VIIa live is has announced that it is about two hours, although this It could vary with different therapeutic embodiments and patients individual. Therefore, the half-life of Factor VIIa in the plasma in a given treatment embodiment should be determined with the Coagulation Assay based on Tissue Factor of poor coagulation

H. Examples

The following examples are included for demonstrate preferred embodiments of the invention. Must be appreciated by those skilled in the art than the techniques described in the following examples they represent techniques discovered by the inventor that work well in the practice of the invention, and so you may consider that they constitute preferred modes for your practice.

Example I Synthesis of truncated tissue factor

The Truncated Tissue Factor in the present description is designated as the extracellular domain of the protein Mature Tissue Factor (amino acids 1-219 of the mature protein; SEQ ID NO: 1). Sequence of identification No. 1 is encoded by, for example, the Identification Sequence No. 10

A. H_ {6} [tTF]

H6 Ala Met Ala [tTF]. The complementary DNA of the Truncated Tissue Factor (cDNA) was prepared as follows: RNA from J-82 cells (from human bladder carcinoma) was used for cloning the Truncated Tissue Factor. Total RNA is isolated using GlassMax MR RNA microinsulation reagent (Gibco BRL). RNA was reverse transcribed into cDNA using the equipment PCR (`` polymerase chain reaction '') of GeneAmp RNA (Perkin Elmer) The truncated Tissue Factor cDNA was amplified using the same team with the following two primers:

5 'primer: 5' GTC ATG CCA TGG CC T CAG GCA CTA CAA (SEQ ID NO: 15)

3 'primer: 5' TGA CAA GCT TA T TCT CTG AAT TCC CCT TTC T (SEQ ID NO: 16)

The underlined sequences encode the N-terminus of the truncated Tissue Factor. The rest of the sequence in the 5 'primer is the restriction site for Nco I allowing the cloning of Truncated Tissue Factor into the expression vector. The sequence in the 3 'primer is the Hin dIII site for cloning of the Truncated Tissue Factor into the expression vector. Polymerase chain reaction amplification was performed as suggested by the manufacturer. In summary, 75 µM dNTP was used; 0.6 µM primer, 1.5 mM MgCl 2 and 30 cycles of 30 seconds at 95 ° C, 30 seconds at 55 ° C and 30 seconds at 72 ° C were performed.

\hbox{(His) _{6} }

en el término N, la cual se puede purificar en una columna Ni-NTA. Además, la proteína de fusión tiene un sitio de disociación de trombina y los residuos 1-219 del Factor de Tejido.Truncated Tissue Factor was expressed as a fusion protein in an unnatural state in E. coli inclusion bodies using the H6 expression vector pQE-60 (Qiagen). The E. coli H6 pQE-60 expression vector was used for the expression of the truncated Tissue Factor (Lee et al., 1994). The truncated Tissue Factor cDNA amplified by polymerase chain reaction was inserted between the Nco I site and Hin dIII. The H 6 pQE-60 has an integrated coding sequence (His) 6 so that the expressed protein has the sequence of

 \ hbox {(His) 6 

in the term N, which can be purified on a Ni-NTA column. In addition, the fusion protein has a thrombin dissociation site and 1-219 residues of Tissue Factor.

To purify the truncated Tissue Factor, the truncated Tissue Factor containing H 6 pQE-60 DNA was transformed into E. coli TG-1 cells. Cells were grown at OD 600 = 0.5 and IPTG was added at 30 µM to induce the production of Truncated Tissue Factor. The cells were harvested after stirring for 18 hours at 30 ° C. The cell pellet was denatured in 6 M Gu-HCl and the lysate was loaded onto a Ni-NTA column (Qiagen). The bound truncated Tissue Factor was washed with 6 M urea and the truncated Tissue Factor was redoubled with a gradient of 6 M - 1 M urea at room temperature for 16 hours. The column was washed with a wash regulator (0.05 Na H 2 PO 4, 0.3 M NaCl, 10% glycerol) and the truncated Tissue Factor eluted with 0.2 M Imidozole in a water regulator washed. The eluted truncated Tissue Factor was concentrated and loaded onto a G-75 column. Truncated Tissue Factor monomers were collected.

B. Truncated Fabric Factor

Gly [tTF]. The complementary DNA of GlytTF (cDNA) it was prepared in the same way as described in the section previous except that the 5 'primer was replaced by the following primer in the polymerase chain reaction.

5 'primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG CTT CTG GCA CTA CAA ATA CT (SEQ ID NO: 17)

The underlined sequence encodes the N-terminus of the truncated Tissue Factor. The remaining sequence encodes a restriction site for Nco I and a dissociation site for thrombin.

The expression vector H 6 pQE60 and the procedure for protein purification is identical to described above except that the final protein product is treated with thrombin to remove the H6 peptide. This was done adding one part of thrombin (Sigma) to 500 parts of Factor of Truncated tissue (weight / weight), and dissociation was carried out at room temperature for 18 hours. The thrombin was removed from the Truncated Fabric Factor by passing the mixture through an affinity column of thrombin Benzamidine Sepharose 6B (Pharmacia). The resulting Truncated Tissue Factor, designated tTF_ {219}, consisted of residues 1-219 of Tissue Factor plus an additional glycine in the term N. This migrated as a single band of molecular weight 26 kDa when analyzed by SDS-PAGE, and the sequence of N-terminal by Edman degradation. Had the Sequence of Identification Sequence No. 1.

C. Truncated Fabric Factors Modified by cysteine

(His) 6 -N'-cys'tTF_ {219} -tTF, abbreviated hereinafter as H_ {6} -N'-cys-tTF_ {219}, was prepared by mutating tTF 219 by chain reaction of polymerase with a 5 'primer encoding a Cys in front of the term N 'of Mature Truncated Tissue Factor. Was prepared equally H_ {6} -tTF_ {219} -cys-C ' using a 3 'primer encoding a Cys after amino acid 219 of the Truncated Fabric Factor. Expression and purification were as for tTF_21, except that the Ellman reagent (acid 5'5'-dithio-bis-2-nitrobenzoic acid) it was applied after redoubling it to convert the Cys N 'or C' terminal in a stable activated disulfide group. The products they had the sequences shown in Identification Sequence No. 2 and Identification sequence No. 3. Thrombin dissociation removed the (His) 6 tag and converted the proteins into N'-cys-tTF_ {219} and tTF_ {219} -cys-C 'having the sequences shown in Identification Sequence No. 4 and Identification sequence No. 5. Products were> 95% cigars judged by gel electrophoresis SDS-polyacrylamide.

H_ {6} -tTF_ {220} -cys-C ' Y H_ {6} -tTF_ {221} -cys-C ' were prepared by mutating tTF 219 by chain reaction of 3 'primers polymerase encoding Ile-Cys e Ile-Phe-Cys after amino acid 219 of the Truncated Tissue Factor. The expression, redouble and purification were as for H_ {6} -tTF_ {219} -cys-C '. Proteins have the sequences shown in Sequence of Identification No. 6 and Identification Sequence No. 7

Example II Synthesis of the dimeric tissue factor

The inventors reasoned that the dimers of Tissue Factor may be more potent than monomers when Start coagulation. It is possible that the Natural Tissue Factor on the surface of bladder carcinoma cells J82 can exist as a dimer (Fair et al., 1987). The link of a Factor VII or Factor VIIa molecule to a Factor Factor molecule Weaving can also facilitate the bonding of another Factor VII or another Factor VIIa to another Tissue Factor (Fair et al., 1987; Bach et al., 1986). In addition, the Fabric Factor shows structural homology to family members receiving cytokine (Edgington et al., 1991) some of which are they dimerize to form active receptors (Davies and Wlodawer, 1995). The inventors therefore synthesized dimers of Factor of Fabric, as follows. Although the synthesis of dimers later in the present description they are described in terms of conjugation chemistry, the inventors also took into account recombinants and other means to produce the dimers herein invention.

A. [tTF] Linker [tTF]

The Gly [tTF] Linker [tTF] was made with the Gly structure [tTF] (Gly) 4 Ser (Gly) 4 Ser (Gly) 4 Ser [tTF]. Two pieces of DNA were amplified with polymerase chain reaction separately and ligated and inserted into the vector as follows:

Polymerase chain reaction 1: Preparation of Truncated Tissue Factor and 5 'half of the linker DNA. The primer sequences in the polymerase chain reaction were as follows:

5 'primer: 5' GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT GCG GCA CTA CAA ATA CT (SEQ ID NO: 18)

3 'primer: 5' CGC GGA TCC ACC GCC ACC AGA TCC ACC GCC TCC TTC TCT GAA TTC CCC TTT CT (SEQ ID NO: 19)

Gly DNA [tTF] was used as the DNA standard. Other conditions of the polymerase chain reaction were as those described in the section of the Truncated Tissue Factor.

Polymerase Chain Reaction 2: The Preparation of the 3 'half of the linker DNA and the Factor DNA Truncated fabric. The primer sequences in the chain reaction Polymerase were as follows:

5 'primer: 5' CGC GGA TCC GGC GGT GGA GGC TCT TCA GGC ACT ACA AAT ACT GT (SEQ ID NO: 20)

3 'primer: 5' TGA CAA GCT TAT TCT CTG AAT TCC CCT TTC T (SEQ ID NO: 21)

Truncated Tissue Factor DNA was used as the standard in the polymerase chain reaction. The product of the polymerase chain reaction 1 was digested with Nco I and Bam H. The polymerase chain reaction product 2 was digested with Hin dIII and Bam H1. The DNA of the polymerase chain reaction 1 and the digested polymerase chain reaction 2 was linked with H 6 pQE 60 DNA digested by Nco I and Hin dIII.

For vector constructions and the protein purification, the procedures were the same as are described in the Gly section [tTF].

B. Cys [tTF] Linker [tTF]

The Cys [tTF] Linker [tTF] was also built with the structure Ser Gly Cys [tTF 2-219] (Gly) 4 Ser (Gly) 4 Ser (Gly) 4 Be [tTF]. DNA was made by polymerase chain reaction using the following primers:

5 'primer: 5'GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT CTT GCG GCA CTA CAA ATA CT (SEQ ID NO: 22)

3 'primer: 5' TGA CAA GCT TAT TCT CTG AAT TCC CCT TTC T (SEQ ID NO: 23)

DNA [tTF] linker [tTF] was used as the standard. The remaining polymerase chain reaction conditions were the same as described in the Fabric Factor section truncated. All vector constructions and purification of proteins were described in the purification of H 6 C [tTF].

C. [tTF] linker [tTF] cys

The dimer [tTF] linker [tTF] was also made cys with the protein structure [tTF] (Gly) 4 Ser (Gly) 4 Ser (Gly) 4 Ser [tTF] Cys. DNA was made by polymerase chain reaction using the following primers:

5 'primer: 5'GTC ATG CCA TGG CCC TGG TGC CTC GTG GTT GCA CTA CAA ATA CT (SEQ ID NO: 24)

3 'primer: 5' TGA CAA GCT TAG CAT TCT CTG AAT TCC CCT TTC T (SEQ ID NO: 25)

[TTF] linker [tTF] DNA was used as the Pattern. The remaining conditions of the chain reaction of polymerase were the same as described in the section of Truncated tissue factor. The vector constructions and the Protein purification were performed again as described in the purification of the section of [tTF] cys.

D. Chemically conjugated dimers

The monomer [tTF] Cys, which had been treated with Ellman's reagent to convert the free Cys into a group activated disulfide, with half a molar equivalent of dithiothreitol. This generated free Cys residues in half of the molecules The monomers chemically conjugated to form dimers [tTF] Cys-Cys [tTF]. This was done by adding a molar amount equal to DTT for the [tTF] Cys protected to room temperature for one hour to protect and expose the cysteine in term C of [tTF] Cys. An equal molar amount of [tTF] Protected Cys was added to the DTT / [tTF] Cys mixture and the incubation continued for 18 hours at room temperature. The dimers were purified on a gel filtration column G-75 Dimers of H_ {6} -tTF_ {220} -cys-C ', H_ {6} -tTF_ {221} -cys-C ' Y H_ {6} -N'-cys-tTF_ {219}. The Cys monomer [tTF] was chemically conjugated to form dimers Using the same method.

Example III Synthesis of tissue factor mutants

Three tissue factor mutants are described truncated that lack the ability to convert Factor VII linked with Truncated Tissue Factor in Factor VIIa. There are 300 times less Factor VIIa in plasma compared to Factor VII (Morrissey et al., 1993). Therefore, the Fabric Factor truncated circulating mutant should be less able to start the coagulation and therefore exhibit very little toxicity. Nevertheless, as soon as the mutant truncated tissue factor has been located in the site of tumor location, as surprisingly demonstrated in the present description, Factor VIIa can be inject to exchange with Factor VII bound with Factor of truncated fabric. The mutated proteins have the sequences shown in Identification Sequence No. 8 and Sequence of identification No. 9 and are active in the presence of the Factor VIIa.

A. [tTF] G164A

The `` [tTF] G164A '' has the structure of mutant protein with amino acid 164 (Gly) of tTF 219 replaced by Ala. The mutagenesis team directed to the site of Chameleon double chain (Stratagene) was used to generate the mutant. The DNA pattern is Gly DNA [tTF] and the primer sequence mutagenizing is:

5 'CAA GTT CAG CCA AGA AAAC (SEQ ID NO: 26)

The G164A mutant is represented by Sequence Identification No. 9. Vector constructions and protein purification procedures described above They were used in the purification of Gly [tTF].

B. [tTF] W158R

Tryptophan at amino acid 158 of tTF 219 was mutated in arginine by MR polymerase chain reaction with a primer encoding this change. The expression, redouble and purification was as for tTF 219. The mutated protein has the sequences shown in Identification Sequence No. 8

C. [tTF] W158R S162A

The [tTF] W158R S162A is a double mutant in which amino acid 158 (Trp) of tTF_21 is replaced by Arg and amino acid 162 (Ser) is replaced by Ala. The same is used mutagenization method than described for [tTF] G164A and [tTF] W158R. The mutagenization primer is:

5 'ACA CTT TAT TAT CGG AAA TCT TCA GCT TCA GGA AAG (SEQ ID NO: 27)

       \ newpage
    

The previous vector constructions and the Protein purification are the same as those used for purify Gly [tTF].

Example IV Preparation of bispecific dE tTF antibody adducts and synthesis of tissue factor conjugates A. Preparation of bispecific antibody adducts for tTF.

Bispecific antibodies were constructed that they had a Fab 'arm of the 10H10 antibody that is specific for a non-inhibitory epitope of truncated Tissue Factor linked to a Fab 'arm of antibodies (OX7, Mac51, CAMPATH-2) of irrelevant specificity. When mixed with Fabric Factor truncated, bispecific antibody binds to Tissue Factor truncated through arm 10H10, forming a non-covalent adduct. Bispecific antibodies were synthesized according to the Brennan et al. method (1985; incorporated herein description by reference) with minor modifications.

In summary, fragments of F (ab ') 2 from IgG antibodies by means of Pepsin digestion (type A; EC 3.4.23.1) and purified until its homogeneity by chromatography on Sephadex G100. I know reduced F (ab ') 2 fragments for 16 hours at 20 ° C with 5 mM 2-mercaptoethanol in 0.1 M regulator of sodium phosphate, pH 6.8, containing 1 mM EDTA (regulator PBSE) and 9 mM NaAsO2. Ellman reagent (ER) was added to give a final concentration of 25 mM and, after 3 hours at 20 ° C, Fab 'fragments derived from Ellman were separated (Fab'-ER) of Ellman reagents on columns of Sephadex G25 in PBSE.

To form the bispecific antibody, Fab'-ER derived from an antibody was concentrated in approximately 2.5 mg / ml in an Amicon ultrafiltration cell and was reduced with 10 mM 2-mercaptoethanol during a hour at 20 ° C. The resulting Fab'-SH was filtered to through a column of Sephadex G25 in PBSE and mixed with a 1: 1 molar excess of Fab'-ER prepared from the second antibody. The mixtures were concentrated by ultrafiltration to about 3 mg / ml and stirred for 16 hours at 20 ° C. The reaction products were fractionated on Sephadex columns G100 in phosphate regulated saline solution. The fractions that containing bispecific antibody (110 kDa) were concentrated at 1 mg / ml, and stored at 4 ° C in 0.02% sodium azide.

To form antibody adducts bispecific of tTF, the bispecific antibody was mixed with a molar equivalent of tTF or derivatives thereof for 1 hour at 4 ° C. The eluted adduct with a molecular weight of approximately 130 kDa in gel filtration columns, corresponding to a molecule of Bispecific antibody was bound with a tTF molecule.

1. Preparation of IgG-H_ {6} -N'-cys-tTF_ {219} and of IgG-H6 -tTF_ {219} -cys-C '

At 26 mg of immunoglobulin G at a concentration 10 mg / ml phosphate saline solution washed with N 2 se They added 250 micrograms of SMPT (Pharmacia) in 0.1 ml of dry DMF. After stirring for 30 minutes at room temperature, it is applied the solution to a column (1.6 cm in diameter X 30 cm) of Sephadex G25 (F) balanced on the same regulator. The Derived immunoglobulin G was collected in a volume of 10 to 12 ml and concentrated to approximately 3.5 ml by ultrafiltration (Amicon, YM2 membrane). The H_ {6} -N'-cys-tTF_ {219} or H_ {6} -tTF_ {219} -cys-C ' (15 mg) was reduced by incubation at room temperature in the presence of 0.2 mM DTT until Ellman substance was released (ie OD at 412 nm reached a maximum). Then it was applied to the Sephadex G25 column (F) (1.6 cm in diameter x 30 cm) balanced with a regulator washed with N2.

Cys-tTF was added (\ sim15 ml) directly to the derived IgG solution. The mixture is concentrated to approximately 5 ml by ultrafiltration and was incubated at room temperature for 18 hours before resolution by gel filtration chromatography in Sephacryl S200 The peak that contained the material that had a molecular weight of 175,000-200,000 was collected. This component it consisted of an IgG molecule linked to one or two molecules of tTF. The conjugates have the structure:

7

2. Preparation of Fab'-H6-N'-cys-tTF219

Fab 'fragments were produced by reduction of F (ab ') 2 fragments of IgG with 10 mM of mercaptoethylamine The resulting Fab 'fragments were separated of the reducing substance by gel filtration on Sephadex G25 The recently reduced Fab 'fragment and the H_ {6} -N'-cys-tTF_ {219} Ellman's modified were mixed in equimolar amounts to a 20 µM concentration. The progress of the reaction of coupling was followed by the increase in absorbency at 412 nm due to anion 3-carboxylate-4-nitrothiophenolate released as a result of conjugation. The conjugate had the structure:

Fab'-SS-tTF

B. Synthesis of the tissue factor conjugates 1. Chemical derivation and antibody conjugation

The tTF antibody conjugates are synthesized by chemically derived antibody binding to the Truncated Tissue Factor chemically derived through a disulfide bond

The antibody was reacted with an excess 5-fold molar of succinimidil oxycarbonyl-? -methyl- ?- (2 -pyridyldithium) toluene (SMPT) for one hour at temperature environment to produce an antibody derived with an average of 2 pyridyldisulfide groups per antibody molecule. Antibody derivative was purified by permeation chromatography in gel.

A 2.5 fold molar excess was reacted of Truncated Tissue Factor on an antibody with a molar excess 45 times of 2-iminothiolane (2IT) for one hour at room temperature to produce Truncated Tissue Factor with an average of 1.5 sulfydryl groups per molecule of Factor Truncated fabric. The derived Truncated Tissue Factor is also purified by gel permeation chromatography and immediately mixed with the derived antibody.

The mixture was allowed to react for 72 hours at room temperature and then a Sefacril column was applied S-300 to separate the conjugate from Antibody-Factor of Truncated Tissue Factor Truncated tissue free and released pyridine-2-thione. The conjugate is separated from the free antibody by affinity chromatography on a column of truncated anti-tissue factor. The predominant molecular species of the final conjugate product was the Conjugate of only substituted antibody-Factor of Truncated Fabric (Mr approximately 176,000) with minors amounts of multiply substituted conjugates (Mr? approximately 202,000) valued by SDS-PAGE.

2. Conjugation of modified Truncated Tissue Factor by cysteine to antibody derived

The conjugates of antibody-C [tTF] and [tTF] C se synthesized by direct coupling of Tissue Factor truncated modified with cysteine to chemically derived antibody through a disulfide bond.

The antibody was reacted with an excess 12 times molar of 2IT for one hour at room temperature to produce derivative antibody with an average of 1.5 groups sulfydryl per antibody molecule. The derived antibody is purified by gel permeation chromatography and immediately mixed with a 2-fold molar excess Factor of Truncated tissue modified with cysteine. The mixture was left react for 24 hours at room temperature and then it purified the conjugate by gel permeation and chromatography by affinity as described above.

The predominant molecular species of the conjugate final was the only substituted conjugate (Mr approximately 176,000) with lower amounts of multiple substituted conjugates (Mr? Approximately 202,000) valued by SDS-PAGE.

3. Conjugation of modified Truncated Tissue Factor with cysteine to Fab 'fragments

Antibody conjugates were prepared Fab'-C [tTF] and [tTF] C. These conjugates they can be more powerful live because they must remain in cell surface for longer than conjugates bivalent due to its limited internalization capacity. The Fab 'fragments are mixed with a 2-fold molar excess of Factor of truncated tissue modified with cysteine for 24 hours and then the conjugate is purified by gel permeation and affinity chromatography as described above.

Example V Tumor infarction by tissue factor A. Methods 1. In vitro coagulation test

This test was used to verify that the Factor of truncated tissue, various derivatives and mutants thereof, and immunoglobulin-tTF conjugates acquire coagulation inducing activity as soon as it is located in a cell surface A20 lymphoma cells were incubated (I-A d positive) (2 X 10 6 cells / ml, 50 mul) for one hour at room temperature with an antibody bispecific (50 micrograms / ml, 25 microliters) consisting of a Fab 'arm of antibody B21-2 directed against I-A d linked with a Fab 'arm of the 10H10 antibody directed against a non-inhibitory epitope on Factor of truncated fabric. The cells were washed at room temperature and variant concentrations of Truncated Tissue Factor were added, derivatives or mutants thereof, or conjugates of immunoglobulin-Tissue Factor truncated during One hour at room temperature. The bispecific antibody captures to the Truncated Tissue Factor or the linked Truncated Tissue Factor with immunoglobulin, putting it in close approximation with the cell surface, where coagulation can proceed.

The cells were washed again at temperature ambient, they were resuspended in 75 microliters of solution phosphate regulated saline and heated to 37 ° C. They were added calcium (12.5 mM) and citrated mouse or human plasma (30 microliters) The time was recorded for the formation of the first strands of fibrin. Coagulation time was plotted with respect to the concentration of Truncated Tissue Factor and they compared the curves with standard curves prepared using standard tTF_ {219} preparations.

In some studies, concentrations were added variants of recombinant human Factor VIIa together with tTF 219 and mutants thereof, to determine if the coagulation capacity was increased by the presence of Factor VIIa.

2. Factor Xa production tests

This assay is useful in addition to or as an alternative to the in vitro coagulation assay to demonstrate that Truncated Tissue Factor and immunoglobulin-tTF conjugates acquire coagulation-inducing activity as soon as they are located on a cell surface. The test measures the conversion rate of Factor X to Factor Xa using a substrate that generates chromophore (S-2765) for Factor Xa.

A20 cells (2 X 10 7) were suspended cells) in 10 milliliters of medium containing 0.2 percent of Weight / volume of sodium azide. To 2.5 ml of cell suspension added 6.8 micrograms of the bispecific `` capture '' antibody B21-2 / 10H10 for 50 minutes at temperature environment. The cells were washed and resuspended in 2.5 ml. of medium containing 0.2 percent weight / volume azide of sodium. Truncated Tissue Factor and conjugates of immunoglobulin-tTF dissolved in the same medium is distributed in volumes of 100 microliters to a range of 96 well microtiter plate concentrations. To the wells were then added 100 microliters of the suspension of bispecific cell / antibody. The plates were incubated for 50 minutes at room temperature.

Plates were centrifuged, discarded supernatants and the cell pellets were resuspended in 250 microliters of wash regulator (150 mM NaCl; 50 mM Tris-HCl, pH 8; 0.2 percent weight / volume of bovine serum albumin). The cells were washed again and the cells were resuspended in 100 microliters of a dilution 12.5 times of Proplex T (Baxter, Inc.) containing factors II, VII, IX and X in dilution regulator (Washing Regulator supplemented with 12.5 mM calcium chloride). The plates are incubated at 37 ° C for 30 minutes. To each well was added stop solution (12.5 mM acid sodium ethylenediamine tetraacetic acid (EDTA) in wash regulator The plates were centrifuged. One hundred microliters of supernatant from each well were added to 11 microliters of S-2765 (Dihydrochloride N-? -Benzyloxycarbonyl-D-Arg-L-Gly-L-Arg-p-nitroanilide, Chromogenix AB, Sweden). The optical density of each solution is measured at 409 nm. The results were compared with standard curves generated from tTF_ 219 standard.

3. Live tumor thrombosis

This model was used to demonstrate that the Factor of Truncated tissue and conjugates of immunoglobulin-tTF induced vessel thrombosis blood tumors and caused the tumor infarction in vivo.

The tumor test systems were four Types: (i) 3LL mouse lung carcinoma growing subcutaneously in C57BL / 6 mice; (ii) mouse neuroblastoma C1300 growing subcutaneously in BALB / c nu / nu mice; (iii) HT29 human colorectal carcinoma growing subcutaneously in BALB / c nu / nu mice; and (iv) C1300 Mu? mouse neuroblastoma growing subcutaneously in BALB / c nu / nu mice. C1300 tumor Muγ is a transfectant that secretes interferon- γ derived from the C1300 tumor (Watanabe et al., 1989).

In addition, the C1300 (Muγ) tumor model of (Burrows et al., 1992; incorporated herein description by reference) was used and modified as follows: (i) B21-2 antibody was used to target I-A d; (ii) C1300 tumor cells were used (Mu γ), a subline of C1300 tumor cells (Mu γ) 12, which grow continuously in BALB / c nu / nu mice; Y (iii) tetracycline was omitted from mice drinking water to prevent gut bacteria from inducing I-A d in the gastrointestinal epithelium. TO unlike immunotoxins, coaguligands and Tissue Factor constructs do not damage the intestinal epithelium which expresses I-A d.

4. Tumor establishment

To establish tumors, they were injected subcutaneously 10 6 to 1.5 x 10 7 tumor cells in the right front flank of the mice. When the tumors of different sizes, mice were randomly assigned to different study groups. Then the mice received a 0.5 mg / kg intravenous injection of Truncated Tissue Factor alone or bound with IgG, Fab ', or bispecific antibody. Other mice received equivalent amounts of IgG, Fab 'or only bispecific antibody. Injections were performed slowly in tail veins for about 45 seconds, usually followed by 200 microliters of saline solution.

In some studies, the effect of administer chemotherapeutic drugs against cancer on the thrombotic action of Truncated Tissue Factor in blood vessels of the tumor To mice that had subcutaneous colorectal tumors human HT29 1.0 cm in diameter were given injections intraperitoneal doxorubicin (1 mg / kg / day), camptothecin (1 mg / kg / day), etoposide (20 mg / kg / day) or gamma interferon (2 x 10 5 units / kg / day) for two days before the injection of Truncated Tissue Factor and again on the day of injection of Truncated tissue factor.

Twenty four hours after being injected with Truncated Tissue Factor or conjugates of Immunoglobulin-Truncated Tissue Factor, the mice were anesthetized with metophane and bled by infusion with heparinized saline. Tumors and tissues were cut normal and immediately set at 3 percent (volume / volume) formalin. Paraffin sections were cut and stained with hematoxylin and eosin. The blood vessels were counted that had open lumens containing erythrocytes and vessels blood vessels containing thrombi. The paraffin sections are cut and stained with hematoxylin and eosin or with scarlet blue of Martius (MSB) trichrome for fibrin detection.

5. Antitumor effects

Accepted animal models were used to determine whether the administration of Truncated Tissue Factor of immunoglobulin-tissue factor conjugates truncated suppressed the growth of solid tumors in mice. The Tumor test systems were: (i) tumors of disease Human Hodgkin L540 growing in SCID mice; (ii) neuroblastoma C1300 Mu [gamma] (which secretes interferon) growing in mice nu / nu; (iii) human non-small cell lung carcinoma H460 growing in nu / nu mice. To establish solid tumors, it 1.5 x 10 7 injected tumor cells subcutaneously into the right front flank of SCID or BALB / c nu / nu mice (Charles River Labs., Wilmingham, MA). When the tumors grew to different diameters, mice were assigned to different groups experimental, each containing 4 to 9 mice.

The mice then received an injection. 0.5 mg / kg intravenous tissue factor truncated alone or linked with bispecific antibody. Other mice received equivalent amounts of bispecific antibody only. The injections were carried out for approximately 45 seconds in one of the tail veins, followed by 200 microliters of Saline solution. The infusions were repeated six days later. I know measured the perpendicular diameters of the tumors at intervals regular and tumor volumes were calculated.

B. Results 1. In vitro coagulation by truncated Tissue Factor and variants

To direct the Truncated Tissue Factor to IA d in vascular endothelium of the tumor, the inventors prepared a bispecific antibody with Fab 'arm of B21-2 antibody, specific for IA d, linked with Fab' arm of the 10H10 antibody, specific for a non-inhibitory epitope in module C of the truncated Tissue Factor. This bispecific antibody, B21-2 / 10H10, measured the truncated Tissue Factor binding in a specific manner for antigen to IA d in B lymphoma cells of A20 mice in vitro . When mouse plasma was added to the A20 cells to which the truncated Tissue Factor had been linked by B21-2 / 10H10, it coagulated rapidly. The fibrin strands were visible 36 seconds after the addition of plasma to the antibody treated cells, compared with 164 seconds when the plasma was added to untreated cells (Figure 4A). Only when the Truncated Tissue Factor was linked to the cells did this increase the coagulation observed: no effect on the coagulation time was seen when the cells were incubated with Truncated Tissue Factor only, with F (ab ') 2, homodimeric, with Fab 'fragments, or with Truncated Tissue Factor plus bispecific antibodies that only had one or two specificities necessary to bind the Truncated Tissue Factor to A20 cells.

TTF_ {219} prepared as in Example I had identical capacity to a preparation of tTF_ {219} `` standard '' obtained from Dr. Thomas Edgington (The Scripps Research Institute, La Jolla, CA) to induce plasma coagulation of mouse or human after binding through antibody Bispecific B21-2 / 10H10 to A20 lymphoma cells (figure 5). Mouse plasma coagulated in 50 seconds when both the preparation of tTF 219 of Example I and the Factor of Standard truncated tissue was applied to the cells at 3 X 10-9 M. Thus, tTF_219 prepared as described in the this description seems to be correctly redoubled and fully active

There was a linear relationship between the logarithm of the number of truncated Tissue Factor molecules bound to the cells and the rate of plasma coagulation by the cells (Figure 4B). In the presence of the cells alone, the plasma coagulated in 190 seconds, while at 300,000 molecules of Tissue Factor truncated per cell the coagulation time was 40 seconds. Even with only 20,000 molecules per cell, coagulation was faster (140 seconds) than with untreated cells. These in vitro studies showed that the thrombogenic potency of Truncated Tissue Factor increases by the proximity of the cell surface mediated through antibody-directed binding to Class II antigens on the cell surface.

H_ {6} -N'-cys-tTF_ {219} Y H_ {6} -tTF_ {219} -cys-C ' they were as active as the Truncated Tissue Factor in inducing the plasma coagulation as soon as they were linked through the antibody bispecific to A20 cells. The plasma coagulated in 50 seconds when they were applied H_ {6} -N'-cys-tTF_ {219} Y H_ {6} -tTF_ {219} -cys-C ' at 3 x 10-9 M, the same concentration as for the Factor of Truncated fabric (figure 5). Thus, the mutation of the Factor of Truncated tissue to enter a sequence (His) 6 and a Cys residue in the term N 'or C' does not reduce its activity coagulation inducer.

H_ {6} -tTF_ {220} -cys-C ', tTF_ {220} -cys-C ', H_ {6} -tTF_ {221} -cys-C ' and tTF_ {221} -cys-C 'were so assets such as tTF_21 to induce plasma coagulation in how much they were located on the surface of A20 cells through the bispecific antibody, B21-2 / 10H10. With all samples at 5 x 10-10 M, the plasma coagulated in 50 seconds (figure 6 and figure 7).

2. In vitro coagulation by truncated Tissue Factor dimers

Dimer H_ {6} -N'cys-tTF_ {219} was so active as the same tTF 219 in inducing plasma coagulation in how much was located on the surface of A20 cells by the bispecific antibody, B21-2 / 10H10. To one 1-2 x 10-10 M concentration, both samples induced coagulation in 50 seconds (figure 8). On the other hand the dimer H_ {6} -tTF_ {221} -cys-C ' It was 4 times less active than the monomer H_ {6} -tTF_ {221} -cys-C ' or tTF_ {219} itself. At a concentration of 4 x 10-9 M, the dimer H_ {6} -tTF_ {221} -cys-C ' induced plasma coagulation in 50 seconds, while the corresponding monomer needed to be applied at 1 x 10-9 M for the same effect on coagulation.

3. Tumor thrombosis in vivo

A histological study was performed to determine if intravenous coaguligand administration B21-2 / 10H10-tTF induced thrombosis selective tumor vasculature in mice that had C1300 (Muγ) subcutaneous neuroblastomas from 0.8 to 1.0 cm diameter (figure 9). Within 30 minutes, all the glasses in everything the tumor thrombosed, containing platelet aggregates occlusive, packed red blood cells, and fibrin. At this time, the tumor cells were histologically indistinguishable from the Tumor cells of untreated mice.

After four hours, however, there were Signs of tumor cell damage. Most of the cells in the tumor separated from each other and had pycnotic nuclei, and the Tumor interstitium commonly contained erythrocytes. About at 24 hours, the tumor showed advanced necrosis, and at 72 hours, the entire central region of the tumor had condensed into a residue amorphous. These studies indicated that the effect predominantly occlusive of the coaguligand of B21-2 / 10H10-tTF in the vessels of the tumor is mediated through the binding of Class II antigens on vascular endothelium of the tumor.

Surprisingly it was noted that there was an action non-specific thrombotic of the discernible truncated Tissue Factor in tumor vessels at later times: in mouse tumors that had been injected 24 hours previously with Factor of Truncated fabric only or Truncated Fabric Factor mixed with bispecific control antibody, OX7 / 10H10, tumors assumed a blackened, bruised appearance beginning in the 30s minutes and being progressively more marked until 24 hours. A histological study revealed that 24 hours after the injection of tTF_ {219} virtually all vessels in all regions of the tumor were thrombosed (figure 9). The vessels contained aggregates of platelets, packed red cells and fibrin. most of the tumor cells had separated from each other and had developed pycnotic nuclei and many regions of tumors They were necrotic. This was more pronounced in the tumor nucleus. Erythrocytes were commonly observed in the interstitium of the tumor.

It is possible that thrombogenic activity resident of the vasculature of the tumor (Zacharski, et al., 1993) make these vessels more susceptible to thrombosis even by Truncated non-directed tissue factor. Alternatively, changes augmented procoagulants could have been induced by the interferon-γ derived from the tumor.

Similar results were obtained when the tTF 219 was administered to mice that had large C1300 tumors (greater than 1000 mm3). Again, virtually all glasses they thrombosed 24 hours after the injection (figure 10). Of this mode, the effects observed in C1300 Muγ tumors are not related to γ interferon secretion by cells Tumor

Other studies were conducted in C57BL / 6 mice that had large 3LL tumors (larger than 800 mm 3). Again, thrombosis of the tumor vessels was observed, although somewhat less pronounced than with the C1300 tumor and the C1300 Muγ tumor. In average 62 percent of 3LL tumor vessels thrombosed (figure 11).

Vessels in C1300 and C1300 Muγ tumors Small (less than 500 mm3) were largely not affected by the administration of tTF_ {219}. In this way, as tumors grow, their susceptibility to thrombosis by tTF_ {219} increases. This is possibly because the cytokines released by tumor cells or cells host that infiltrate the tumor activate the vascular endothelium of the tumor, inducing procoagulant changes in the vessels.

The coaguligand treatment was well tolerated, the mice did not lose weight and retained normal appearance and normal activity levels At the treatment dose of 0.6 mg / kg of B21-2 / 10H10 plus 0.5 mg / kg of Factor Truncated tissue, toxicity was observed in only two out of forty mice (tail vein thrombosis). It is important to note that neither thrombi nor histological or morphological abnormalities were visible in paraffinic sections of liver, kidney, lung, intestine, heart, brain, adrenal, pancreas, or spleen of the mice that had tumors 30 minutes or 24 hours after coaguligand administration or Free Truncated Tissue Factor. In addition, no signs of toxicity were observed (changes in behavior, physical signs, weight changes) in animals treated.

4. Antitumor effects

The inventors immediately investigated whether the intravenous coaguligand administration B21-2 / 10H10-tTF could inhibit the growth of large tumors (0.8 to 1.0 cm in diameter) in mice. The combined results of three separate studies indicate that the mice that received clotting B21-2 / 10H10-tTF had regressions tumor tumors that lasted four months or more. These effects antitumor were significantly higher than for other groups of treatment (figure 12A).

Surprisingly, the inventors they found that the coaguligand antitumor effect B21-2 / 10H10-tTF was attributable, in part, to a non-directed effect of the Truncated Tissue Factor. The tumors in mice that received Truncated Tissue Factor alone or mixed with bispecific control antibodies (CAMPATH II / 10H10 or B21-2 / OX7) grew significantly more slowly than tumors in mice that received antibodies or only saline solution (figure 12A; figure 12B).

Mice that had C1300 Muγ tumors small (300 mm3) were injected intravenously with 16-20 micrograms of tTF 219. The treatment is repeated a week later. The first treatment with tTF_ {219} had a slight inhibitory effect on tumor growth, consistent with the lack of marked thrombosis observed with small anterior tumors (figure 12B). The second treatment had a substantially greater effect on tumor growth, statistically significant (P less than 0.01), probably because the tumors were enlarged. A week later of the second treatment with tTF 219, the tumors had 60 percent of the size of the tumors in the mice that received diluent only. The greatest effectiveness of the second injection it probably derives from the greater thrombotic action of tTF_21 on the vessels in large tumors, noted above.

Similar antitumor effects were observed in the mice that had human lung carcinomas H460 (figure 13). The first treatment with tTF 219 was given when the tumors were small (250 mm3) and had little effect on the rate of increase. The second treatment with tTF_21 was given when the tumors were larger (900 mm3) and caused the tumors they will return to 550 mm3 before growing again.

The antitumor effects were also observed in mice that had human HT29 colorectal carcinomas (figure 14). Nu / nu mice that had large tumors (1200 mm 3) in their flanks were injected intravenously with tTF 219 or solution phosphate regulated saline (control), and growth was monitored of tumors every day for ten days. Tumors in the mice treated with tTF 219 discontinued growth for about 7 days after treatment, while tumors in mice treated with phosphate regulated solution They continued to grow without obstacles.

In animals that did not show complete regression of the tumor after coaguligand treatment B21-2 / 10H10-tTF, tumors they grew again from a surviving microscopic ring of cells in the periphery of the tumor. The immunohistochemical test of these tumors revealed that the vascular endothelium at the invasive edge of the tumors lacked detectable Class II antigens consistent with a lack of thrombosis of these vessels allowing the coaguligating the cell survival of the local tumor. In this way, co-administration of a drug that acts on the same cells Tumors would probably improve efficacy, as noted with another antivascular therapy (Burrows and Thorpe, 1992; Burrows and Thorpe 1993; Burrows and Thorpe 1994; U.S.A. Patent Application numbers 07 / 846,349; 08 / 205,330; 08 / 295,868; and 08 / 350,212).

The inventors previously demonstrated that a Powerful cytotoxic ricin A chain immunotoxin directed against the same tumor cells virtually lacked antitumor activity when administered to mice with tumors large C1300 (Mu γ) (Burrows and Thorpe, 1993; Requests for U.S.A. numbers 07 / 846,349; 08 / 205,330; 08 / 295,868; Y 08 / 350.212). The lack of activity was due to the inability of the immunotoxin having access to mass tumor cells large tumors, serving as witnesses to effectiveness comparison of coaguligand therapy.

Studies using coaguligands confirmed the therapeutic potential of the selective initiation of the cascade of blood coagulation in the tumor vasculature (Requests for U.S.A. numbers 08 / 273,567; 08 / 482,369; 08 / 485,482; 08 / 487,427; 08 / 479,733; 08 / 472,631; 08 / 479,727; and 08 / 481,904). The induction of tumor infarction by directing a thrombogen to the tumor endothelial cell markers is, therefore, a effective anticancer strategy and may still result in the eradication of primary solid tumors and metastases vascularized

The successful use of Truncated Tissue Factor alone or immunoconjugates of Tissue Factor truncated with an antibody of irrelevant specificity initially was a surprising result of the directed studies. Although the mice they received only Truncated Tissue Factor had no regressions of complete tumor, it is clear that the amazing antitumor activity of the Truncated Tissue Factor returns to it and its derivatives of Functionally related tissue factor useful in the solid tumor treatment. The benefits of these compositions as detailed in the present description are of more scope and include the lack of side effects of using these Fabric Factors In addition, it is well within the experience of technicians in the field produce the type of compositions of Truncated Tissue Factors presented in the present invention. These compositions can then be used in the treatment of solid tumors alone or in combination with other solid substances anti-cancer

Example VI Mouse plasma coagulation by conjugates of immunoglobulin-tissue factor

The conjugate IgG-H_ {6} -N'-cys-tTF_ {219} was active to induce mouse plasma coagulation when it located on the surface of A20 cells by means of the antibody Bispecific, B21-2 / 10H10. Induced coagulation in 50 seconds when applied to tissue factor concentrations truncated 5 x 10 -9 M compared to 1 x 10 -9 M for tTF_ {219} and H_ {6} -N'-cys-tTF_ {219} unconjugated (figure 15). Coagulation inducing activity of the conjugate IgG-H_ {6} -N'-cys-tTF_ {219} therefore it was reduced 5 times in relation to the H_ {6} -N'-cys-tTF_ {219} or the tTF_ {219} itself unconjugated.

The slight reduction in conjugation of immunoglobulin G (IgG) could be because the fraction of immunoglobulin G of IgG-H_ {6} -N'-cys-tTF_ {219} prevents access of bispecific antibody B21-2 / 10H10 to the tissue factor fraction truncated (i.e. an aberrant reduction related to the testing method). Probably not because the fraction of immunoglobulin G of IgG-H_ {6} -N'-cys-tTF_ {219} interfere with the formation of the initiation complexes of coagulation because, in previous work, the inventors have found that the fraction of tissue factor truncated in a analog construction, B21-2 IgG-H6 -N'-cys-tTF_ {219}, it is active as the Truncated Tissue Factor is linked through from B21-2 / 10H10 to I-A d antigens in A20 cells (figure 16). Similarly, B21-2 IgG-H6 -tTF_ {219} -cys-C ' It was as active in inducing coagulation as conjugation of N'-linked (Figure 16).

The ability to IgG-H_ {6} -N'-cys-tTF_ {219} and Fab'-H_ {6} -N ' -cys-tTF_ {219} to convert Factor X to Xa into presence of Factors II, VII and IX, once located in the A20 lymphoma cell surface by antibody Bispecific B21-2 / 10H10. Construction Fab'-tTF was as active as the same H_ {6} -N'-cys-tTF_ {219}  to induce the formation of Xa. Construction IgG-tTF was slightly (2 times) less active than the same H_ {6} -N'-cys-tTF_ {219} (figure 17).

Example VII Growth inhibition of C1300 Muγ tumors by immunoglobulin-tissue factor conjugate

Mice with small tumors (300 mm3) C1300 Mu [gamma] subcutaneous were treated with tTF_21 or a complex of tTF 219 and a bispecific antibody, OX7 Fab '/ 10H10 Fab', not directed to a component of the tumor environment. The treatment was repeated six days later (figure 18). The bispecific antibody was simply designed to increase mass of tTF_ {219} from 25 kDa to 135 kDa, and thus prolong its half-life circulatory, and it was not intentional to impart a function target the Truncated Fabric Factor.

Tumors in mice treated with conjugate of immunoglobulin-tTF grew more slowly than those mice that received only tTF 219. Fourteen days after the first injection, the tumors had 55 percent about the size of those in the controls they received only diluent In mice that received tTF 219 alone, the tumors were 75 percent of the size in controls they received diluent only (figure 18).

Example VIII Increased conjugate antitumor activity of immunoglobulin-Tissue Factor truncated by etoposide

Mice that had tumors of the L540 human Hodgkin's disease with a complex of tTF 219 and a bispecific antibody together with the anticancer drug conventional, etoposide. Etoposide greatly increased the immunoglobulin-tTF conjugate action. In this tumor model alone, mice that received the complex of tTF antibody only showed little reduction in tumor growth in relation to tumors in mice that they received only diluent (figure 19).

Instead, tumors in mice that received both the etoposide and the conjugate of immunoglobulin-tTF decreased in size and not They restarted their growth for seventeen days. At the end of study (day 20), tumors in mice that received etoposide plus immunoglobulin-tTF had an average 900 mm 3 volume compared to 2300 mm 3 in the mice treated with diluent and 2000 mm 3 in treated mice with only immunoglobulin-tTF. In mice that they received only etoposide, the tumors averaged 1400 mm3 on day 14 (figure 19). These results indicate that the Etoposide can predispose tumor vessels to thrombosis by tTF or immunoglobulin-tTF conjugates. Regardless of the mechanism, the results clearly show that the advantageous combination of Tissue Factor, or a conjugate of Tissue Factor with a classic chemotherapeutic substance.

Example IX Increased plasma coagulation by VIIa

The capacity of tTF_ {219} associated with the cell to induce mouse or human plasma coagulation increased much with the presence of the free factor VIIa (figure 20). In absence of Factor VIIa, A20 cells treated with antibody bispecific B21-2 / 10H10 and 10-10 M of tTF_ {219} coagulated the plasma in 60 seconds, while in presence of 13.5 nM Factor VIIa, coagulated the plasma in 20 seconds (figure 20). This represents approximately an increase in 100 times the coagulation induction power of tTF in presence of Factor VIIa. Still in the presence of 0.1 nM Factor VIIa, a 2-5 fold increase in power was observed Coagulation induction factor truncated.

This finding leads to aspects of the description that refer to the co-administration of Factor VIIa together with Truncated Fabric Factor or derivatives thereof, or with immunoglobulin-tissue factor conjugates truncated, in order to increase thrombosis of the tumor vessels Live.

Example X Reduced coagulation of mouse plasma by mutants of Tissue Factor Activation Factor VII

Mutations in W158 and G164 have been released of tTF_ {219} to significantly reduce the capacity of the Factor of Tissue to induce coagulation of recalcified plasma (Ruf and collaborators, 1992; Martin et al., 1995). Waste from 157-167 of the Tissue Factor appear to be important to accelerate the activation of Factor VII in Factor VIIa, but not the binding of Factor VII to the Tissue Factor. The inventors mutated W158 to R and G164 to A and determined whether the mutants acquired the ability to coagulate plasma as soon as they were located through a bispecific antibody, B21 / 2-10H10, in the A20 cell surface. It was found that the mutants were 30 at 50 times less effective than what tTF_ {219} was in inducing plasma coagulation (figure 21).

Example XI Restoration of the coagulation capacity of mutants of Factor VII activation by Factor VIIa

The tTF_21 mutant (G164A) is a very mutant weakly coagulant of tTF 219 (Ruf, et al., 1992). The mutation is present in a region of the Tissue Factor (amino acids 157-167) thought to be important for the conversion of Factor VII to Factor VIIa. In this way, the addition of Factor VIIa to antibody coated cells bispecific and tTF_ {219} (G164A) would be reasoned to induce the plasma coagulation In support of this, A20 cells coated with B21-2 / 10H10 followed by tTF_ {219} (G164A) had increased capacity to induce plasma coagulation in the presence of Factor VIIa (figure 22). The addition of Factor VIIa at 1 nM or more produced only marginally coagulation times slower than that observed with tTF_ {219} and Factor VIIa in the same concentrations.

The tTF_21 mutant (W158R) gave results similar to tTF_ {219} (G164A). Again, in addition to the Factor VIIa at 1 nM or more to A20 cells coated with B21-2 / 10H10 followed by tTF_21 only gave coagulation times marginally slower than tTF_ {219} and Factor VIIa at the same concentrations.

These results support aspects of the description that discloses that tTF_ {219} (G164A) or tTF_ {219} (W158R), when animals that are co-administered with Factor VIIa they have a tumor, they will induce thrombosis of the tumor vessels. East approach is considered to be advantageous because tTF (G164A), tTF (W158R) or Factor VIIa given separately are practically not toxic to mice, and the same is reasonably expected in humans. Co-administration of the mutant tTF and Factor VIIa is expected not to cause toxicity, and cause efficient thrombosis of Tumor vessels Administration of Truncated Tissue Factor mutant along with Factor VIIa is taken into account so it gives as result an improved therapeutic index in relation to the tTF_ {219} plus Factor VIIa.

Example XII Increased antitumor activity of activation mutants and Factor VIIa

For these studies, the inventors chose the HT29 xenograft tumor model (human colorectal carcinoma). HT29 cells (10 7 cells / mouse) were injected subcutaneously in BALB / c nu / nu mice. The size of the tumor and animals were treated when the tumor size was 0.5 and 1.0 cm3. The animals were given an injection IV of one of the following tTF_ {219} (16 \ mug), tTF_ {219} (16 \ mug) + Factor VIIa (1 \ mug), tTF_ {219} (G164A) (64 \ mug), tTF_ {219} (G164A) (64 \ mug) + Factor VIIa (1)), Factor VIIa alone (1 mug), or saline.

The animals were sacrificed 24 hours later of the treatment, they were flooded with saline and heparin and they bled Tumors and organs were collected, fixed with formalin and histological sections were prepared. It was quantified the average area of necrosis in sections of the tumor and was calculated as a percentage of the total area of the tumor in the section.

In these small HT29 tumors, analysis of the tumor sections of animals treated with saline, Factor VIIa, tTF21 or tTF21 (G164A) showed some necrosis (Figure 23). The tumor necrosis induced by tTF was the most developed, although this was not surprising, this time, as the results of previous studies using different tumor models and / or larger tumors. An analysis of the tumor sections of animals treated with tTF 219 + Factor VIIa or tTF 219 (G164A) + Factor VIIa revealed considerable necrosis (12.5 percent and 17.7 percent respectively; Figure 23) and a greater correlation between recently thrombosed blood vessels and areas of necrosis. The combined use of Factor VIIa with Tissue Factor, even a Tissue Factor construction with particularly poor in vitro coagulant activity, is therefore a particularly advantageous aspect of the present invention. Since the HT29 tumor model is difficult to thrombus in general and these tumors were small in size, these results probably translate to more shocking results in other systems and in humans.

References

Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory , 1988 .

Abraham et al., Science , 233: 545-548, 1986 .

Abrams and Oldham , Monoclonal Antibody Therapy of Human Cancer , Foon and Morgan (Eds.), Martinus Nijhoff Publishing, Boston, pp. 103-120, 1985 .

Ausubel et al., Current Protocols in Molecular Biology , Greene Publishing Associates and Wiley Interscience, NY, 1989 .

Bach et al., Biochemistry , 25, 4007-4020, 1986 .

Bauer , et al., Vox Sang , 61-156-157, 1991 .

Baxter , and collaborators, Micro. Res ., 41 (1): 5-23, 1991 .

Bevilacqua , and collaborators, Proc. Natl Acad. Sci. USA, 84: 9238-9242, 1987 .

Bhagwat et al., Nature , 316: 511-513, 1985 .

Bicknell and Harris , Seminars in Cancer Biology , 3: 399-407, 1992 .

Birembaut et al., J. Pathology , 145: 283-296, 1985 .

Bjorndahl et al., Eur. J. Immunol ., 19: 881-887, 1989 .

Bolhuis et al., J. Immunol ., 149: 1840-1846, 1992 .

Borden et al., Cancer , 65: 800-814, 1990 .

Brennan et al., Science , 229: 81-83, 1985 .

Brinkmann et al., Proc. Natl Acad. Sci ., 88 (19): 8616-8620, 1991 .

Broze , Seminars in Hematol ., 29: 159-169, 1992 .

Burchell et al., J. Immunol ., 131 (1): 508-513, 1983 .

Burrows et al., Cancer Res ., 52: 5965-5962, 1992 .

Burrows et al., Cancer Res ., 51: 4768-4775, 1991 .

Burrows and Thorpe , Proc. Natl Acad. Sci ., USA, 90: 8996-9000, 1993 .

Burtin et al., Cancer , 31: 719-726, 1983 .

Byers and Baldwin Immunol ., 65: 329-335, 1988 .

Campbell , In: Monoclonal Antibody Technology, Laboratory Techniques in Biochemistry and Molecular Biology , Vol. 13, Burden and Von Knippenberg (Eds.), Elseview, Amsterdam, pp. 75-83, 1984 .

Chen et al., J. Immunol ., 145: 8-12, 1990 .

Cherwinski et al., J. Exp. Med ., 166: 1229-1244, 1989 .

Clark et al., Biochem. Biophys ACTA , 867: 244-251, 1986 .

Clark et al., Int. J. Cancer , 2: 15-17, 1988 .

Clark et al., Cancer Res ., 51: 944-948, 1991 .

Colcher et al., Cancer Res ., 47: 1185 and 4218, 1987 .

Collins et al., Proc. Natl Acad. Sci . USA, 81: 4917-4921, 1984 .

Cotran et al., J. Exp. Med ., 164: 661-666, 1986 .

Daar et al., Transplantation , 38 (3): 293-298, 1984 .

Davie et al., Biochem ., 30: 10363-10310, 1991 .

Davies and Wlodawer , FASEB J., 9: 50-56, 1995 .

Davis and Preston , Analytical Biochemistry , 116 (2): 402-407, 1981 .

DeFranco , Nature , 352: 754-755, 1991 .

deLeij et al., Bispecific Antibodies and Targeted Cellular Cytotoxicity , Romet-Lemonne et al., p. 249, 1991 .

Denekamp et al., Brit. J. Cancer , 461: 711-720, 1982 .

Dewerchin et al., Blood , 78 (4): 1005-1018, 1991 .

Di Scipio et al., Biochemistry , 16: 5253-5260, 1977 .

Dillman et al., Antibody Immunocon. Radiopharm ., 1: 65-77, 1988 .

Drake et al., J. Cell Biol ., 109: 389-95, 1989 .

Dustin et al., J. Immunol ., 137: 245-254, 1986 .

Dvorak et al., J. Exp. Med ., 174: 1275-1278, 1991 .

Edgington et al. Thrombosis and Haemostatis , 66 (1): 67-79, 1991 .

Embleton et al., Br. J. Cancer , 63 (5): 670-674, 1991 .

Epenetos et al., Cancer Res ., 46: 3183-3191, 1986 .

Fair , Blood, 62: 784-791, 1983 .

Fair et al., J. Biol. Chem ., 262, 11692-11698, 1987 .

Ferrara , J. Cell. Biochem ., 47: 211-218, 1991 .

Flavell et al., Br. J. Cancer , 64 (2): 274-280, 1991 .

Flavell et al., Br. J. Cancer , 65: 545-551, 1992 .

Folkman , Adv. Cancer Res ., 43: 175-230, 1985a

Folkman , In: Important Advances in Oncology , Part I, DeVita et al., (Eds.), JB Lippincott, Philadelphia, pp. 42-62, 1985b .

Fox et al., J. Biol. Resp ., 9: 499-511, 1990 .

Frelinger III, et al., J. Biol. Chem ., 265 (11): 6346-6352, 1990 .

Frelinger III, et al., J. Biol. Chem ., 266 (26): 17106-17111, 1991 .

French et al., Cancer Res ., 51: 2358-2361, 1991 .

Galfre et al., Methods Enzymol ., 73: 1-46, 1981 .

Gefter et al., Somatic Cell Genet ., 3: 231-236, 1977 .

Geppert et al., Immunological Reviews , 117: 5-66, 1990 .

Ghose and Blair , CRC Critical Reviews in Therapeutic Drug Carrier Systems , 3: 262-359, 1987 .

Ghose , CRC Critical Review in Therapeutic Drug Carrier Systems , 3: 262-359, 1982 .

Ghose et al., Meth. Enzymology , 93: 280-333, 1983 .

Ghose et al., CRC Critical Reviews in Therapeutic Drug Carrier Systems , 3: 262-359, 1987 .

Giles et al., Brit. J. Haematol ., 69: 491-497, 1988 .

Glennie et al., J. Immunol ., 139: 2367-2375, 1987 .

Glennie et al., 1988 .

Goding , In: Monoclonal Antibodies: Principles and Practice , 2d Ed., Academic Press, Orlando, Fl., Pp. 60-61, 65-66, 71-74, 1986 .

Gougos and Letarte , J. Immunol ., 141: 1925-1933, 1988 .

Groenewegen et al., Nature , 316: 361-363, 1985 .

Hagen et al., Proc. Natl Acad. Sci . USA, 83: 2412-2416, 1986 .

Halling et al., Nucl. Acids Res ., 13: 8019-8033, 1985 .

Hammerling , Transplant Rev. , 30: 64-82, 1976 .

Hattey et al., Thrombosis Research , 45 (5): 485-495, 1987 .

Hayward et al., Biological Chemistry , 266 (11): 7114-7120, 1991 .

Hess et al., Transplantation , 6: 1232-1240, 1991 .

Heynen et al., J. Clin. Invest ., 94: 1098-1112, 1994 .

Jain , Cancer Meta. Rev. , 9 (3): 253-266, 1990 .

June et al., Mol. Cell Biol ., 12: 4472-4481, 1987 .

June et al., Immunology Today , 11 (6): 211-216, 1990 .

Juweid et al., Cancer Res ., 52: 5144-5153, 1992 .

Kandel et al., Cell , 66: 1095-1104, 1991 .

Kim et al., Nature , 362: 841-844, 1993 .

Kimura et al., Immunogenetics , 11: 373-381, 1983 .

Kisiel , J. Biol. Chem ., 254 (23): 12230-12234, 1979 .

Klagsburn and Folkman , Angiogenesis Handbook of Experimental Pharmacology , Vol. 95, Sporn and Roberts, Springer-Verlag, Berlin, pp. 549-586, 1990 .

Kohler and Milstein , Nature , 256: 495-497, 1975 .

Kohler and Milstein , Eur, J. Immunol ., 6: 511-519, 1976 .

Krishnaswamy et al., J. Biol. Chem ., 267: 26110-26120, 1992 .

Kyte and Doolittle , J. Mol. Biol ., 157 (1): 105-132, 1982 .

Lamb et al., Eur. J. Biochem ., 148: 265-270, 1985 .

Lee et al., Methods in Enzymology , 237: 146-164, 1994 .

Leith et al., British J. Cancer , 66 (2): 345-348, 1992 .

Lord et al., In: Genetically Engineered Toxins , Frank (Ed.), M. Dekker Publ., P. 183, 1992 .

Lowder et al., Blood , 69: 199-210, 1987 .

Lowe et al., Immunol. Lett ., 12: 263-269, 1986 .

Maeda et al., J. Invest. Derm ., 97: 183-189, 1991 .

Manabe et al., J. Lab. Clin. Med ., 104 (3): 445-454, 1984 .

Martin , FASEB J. , 9: 852-859, 1995 .

Massoglia et al., J. Cell. Phys ., 132: 531-537, 1987 .

Mazzocchi et al., Cancer Immunol. Immuother ., 32: 13-21, 1990 .

Mignatti et al., J. Cell. Biol ., 113: 1193-1201, 1991 .

Miotti et al., Cancer Res ., 65: 826, 1985 .

Moroi and Aoki , J. Biol. Chem ., 251 (19): 5956-5965, 1976 .

Morrissey et al., Cell , 50: 129-135, 1987 .

Morrissey et al., Thrombosis Research , 52: 247-261, 1988 .

Morrissey et al., Blood , 81: 734-744, 1993 .

Mueller et al., Proc. Natl Acad. Sci . USA, 89: 11832-11836, 1992 .

Murray , Clauss , Thurston , Stern , Int. J. Radiat. Biol ., 60: 273-277, 1991 .

Nakamura , Prog. Growth Factor Res ., 3: 67-86, 1991 .

Nawroth , Handley , Matsueda , de Waal , Gerlach , Blohm , Stern , J. Exp. Med ., 168: 637-648, 1988 .

Nawroth and Stern , J. Exp. Med ., 163: 740-745, 1986 .

Nelson , Cancer Cells , 3 (5) pl63-72, 1991 .

Nemerson , Blood , 71 (1): 1-8, 1988 .

Nitta et al., Lancet , 335: 368-371, 1990 .

Nolan and Kennedy , 1990 .

O'Brien et al., J. Clin. Invest ., 82: 206-211, 1988 .

O'Connell et al., J. Immunol ., 144 (2): 521-525, 1990 .

O'Hare et al., FEBS Lett ., 210: 731, 1987 .

Ogata , J. Biol. Chem ., 256: 20678-20685, 1990 .

Ogawa , Shreeniwas , Brett , Clauss , Furie , Stern , Brit. J. Haematol ., 75: 517-524, 1990 .

Ohuchida et al., J. Am. Chem. Soc. , 103 (15): 4597-4599, 1981 .

Oi and Morrison , Mt. Sinai J. Med ., 53 (3): 175-180, 1986 .

Osborn et al., Cell , 59: 1203-1211, 1989 .

Osterud et al., Thrombosis Res ., 42: 323-329, 1986 .

Paborsky et al., J. Biol. Chem ., 266 (32): 21911-21916, 1991 .

Palleroni et al., Int. J. Cancer , 49: 296-302, 1991 .

Pasqualini et al., Nat. Biotechnol . 15: 542-546, 1997 .

Paulus , 1985 .

Perez et al., Nature , 316: 354-356, 1985 .

Perez et al., J. Immunol ., 137: 2069-2072, 1986a .

Perez et al., J. Exp. Med ., 163: 166-178, 1986b .

Pieterez et al., Antibody Immunoconj. Radiopharm ., 1: 79-103, 35, 1988 .

Pimm et al., J. Cancer Res. Clin. Oncol ., 118: 367-370, 1992 .

Pober et al., J. Exp. Med ., 157: 1339-1353, 1991 .

Pukrittayakamee et al., Mol. Biol. Med ., 1: 123-135, 1983 .

Qian et al., Cancer Res ., 140: 3250, 1991 .

Rehemtulla and collaborators, Thrombosis and Haemostatis . 65 (5): 521-527, 1991 .

Reisfeld et al., Melanoma Antigens and Antibodies , p. 317, 1982 .

Remington's Pharmaceutical Sciences , 16th Ed., Mack Publishing Company, 1980 .

Rettig et al., Proc. Natl Acad. Sci . USA., 89: 10832-10836, 1992 .

Rivoltini et al., 3rd Int. Conf. Bispecific Antibodies and Targeted Cellular Cytotoxicity , 1992 .

Ruco et al., Am. J. Pathol ., 137 (5): 1163-1171, 1990 .

Ruf and Edgington , Proc. Natl Acad. Sci . USA., 88: 8430-8434, 1991a .

Ruf and Edgington , Thrombosis and Haemostasis , 66 (5): 529-533, 40, 1991b .

Ruf , et al., J. Biol. Chem. , 266, pg. 2158, 1991 .

Ruf and Edgington , FASEB J. , 8: 385-390, 1994 .

Ruf et al., J. Biol. Chem ., 267: 22206-22210, 1992a .

Ruf et al., J. Biol. Chem ., 267: 6375-6381, 1992b .

Ruf et al, J. Biol Chem, 267 (31):.. 22206-22210, 1992c.

Sakai and Kisiel , Thrombosis Res ., 60: 213-222, 1990 .

Sambrook et al., Molecular Cloning, A Laboratory Manual , Cold Spring Harbor Laboratory, NY, 1989 .

Sands , Immunoconjugates and Radiopharmaceuticals , 1: 213-226, 1988 .

Schutt et al., Immunol. Lett ., 19: 321-328, 1988 .

Shen and Tai , J. Biol. Chem ., 261 (25): 11585-11591, 1986 .

Segal et al., Int. J. Cancer Suppl ., 7 p36-8, 1992 .

Shankar et al., J. Biol. Chem ., 269 (19): 13936-13941, 1994 .

Shepard et al., J. Clin Immunol ., 11: 117-127, 1991 .

Shockley et al. Ann NY Acad. Sci ., 617: 367-382, 1991 .

Smith et al., Br. J. Cancer , 59 (2) p174-8, 1989 .

Spiegelberg and Weigle , J. Exp. Med ., 121: 323-338, 1965 .

Staerz et al., Nature , 314 (6012): 628-631, 1985 .

Stevenson et al., Chem. Immunol ., 48: 126-166, 1990 .

Stone , et al., Biochem J. , 310: 605, 1995 .

Street et al., Cell. Immunol ., 120: 75-81, 1989 .

Sugama et al., Jpn. J. Pharmacol ., 55: 2, pp. 287-290, 1991 .

Sugama et al., J. Cell. Biol 119 (4): 935-944, 1992 .

Ten Cate et al., J. Clin. Invest ., 92: 1207-1212, 1993 .

Thieme et al., Diabetes , 44 (1): 98-103, 1995 .

Thor et al., Cancer Res ., 46: 3118, 1986 .

Ting et al., J. Immunol ., 141: 741-748, 1988 .

Titus et al., J. Immunol ., 138: 4018-4022, 1987 .

Tomiyama et al., Blood ., 79 (9): 2303-2312, 1992 .

Tone 1977 .

Tutt et al., Eur. J. Immunol ., 21: 1351-1358, 1991 .

U.S.A. number 4,196,265

U.S.A. number 4,554,101

U.S.A. number 4,975,369

U.S.A. number 5,017,556

U.S.A. number 5,110,730

U.S.A. number 5,139,941

U.S.A. number 5,183,756

U.S.A. number 5,223,427

U.S.A. number 5,242,813

U.S.A. number 5,288,641

U.S.A. number 5,346,991

U.S.A. number 5,374,617

U.S.A. number 5,437,864

U.S.A. number 5,504,064

U.S.A. number 5,504,067

U.S.A. number 5,589,173

U.S.A. number 5,589,363

Ugarova et al., J. Biol. Chem ., 268 (28): 21080-21087, 1993 .

Vaickus et al., Cancer Invest ., 9: 195-209, 1991 .

Van Duk et al., Int. J. Cancer , 43: 344-349, 1989 .

Venkateswaran et al., Hybridoma , 11 (6): 729-739, 1992 .

Vitetta et al., Cancer Res ., 15: 4052-4058, 1991 .

Wang et al., Biochem and Biophys. Res. Comm ., 177 (1): 286-291, 1991 .

Wang et al., Int. J. Cancer , 54: 363-370, 1993 .

Warr et al., Blood , 75: 1481-1489, 1990 .

Watanbe et al., Proc. Natl Acad. Sci . USA, 86: 9456-9460, 1989 .

Wawrzynczak and Thorpe In : Immunoconjugates: Antibody Conjugates in Radioimaging and Therapy of Cancer , Vogel (ed.), New York, Oxford University Press, pp. 28-55, 1987 .

Weiss et al., Blood , 73: 968-975, 1989 .

Whittle et al., Nature , 292: 472-474, 1981 .

Wildgoose et al., Blood , 80: 25-28, 1992 .

Williams and Esnouf , Biochem. J. , 84: 52-62, 1962 .

Wiman and Collen , Eur. J. Biochem ., 78: 19-26, 1977 .

Wiman , Biochem. J. , 191: 229-232, 1980 .

Winter and Milstein , Nature , 349: 293-299, 1991 .

International Patent Application number WO 94/05328.

International Patent Application number WO 94/07515.

International Patent Application number WO 94/28017.

International Patent Application number WO 94/01653.

Wu et al., Int. J. Pharm ., 12: 235-239, 1990 .

Xu et al., J. Biol. Chem ., 267 (25): 17792-17803, 1992 .

Yamaue et al., Biotherapy , 2: 247-259, 1990 .

Zamarron et al., J. Biol. Chem ., 266 (24): 16193-16199, 1991 .

         \ vskip0.333000 \ baselineskip
      

<110> BOARD OF REGENTS, THE UNIVERSITY OF TEXAS

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<120> METHODS OF FACTORS OF FABRICS AND COMPOUNDS FOR COAGULATION AND TUMOR TREATMENT

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<130> 4001: 001910

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<140> PCT / US98 / 01012

         \ vskip0.333000 \ baselineskip
      

<141> 1998-01-20

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<150> US 60 / 042,427

         \ vskip0.333000 \ baselineskip
      

<151> 1997-03-27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<150> US 60 / 036,205

         \ vskip0.333000 \ baselineskip
      

<151> 1997-01-27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<150> US 60 / 035,920

         \ vskip0.333000 \ baselineskip
      

<151> 1997-01-22

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<160> 27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<170> PatentIn Ver. 2.0

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 1

         \ vskip0.333000 \ baselineskip
      

<211> 219

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 1

8

9

         \ vskip0.333000 \ baselineskip
      

<210> 2

         \ vskip0.333000 \ baselineskip
      

<211> 234

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 2

10

eleven

         \ vskip0.333000 \ baselineskip
      

<210> 3

         \ vskip0.333000 \ baselineskip
      

<211> 234

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 3

12

13

         \ vskip0.333000 \ baselineskip
      

<210> 4

         \ vskip0.333000 \ baselineskip
      

<211> 220

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 4

14

         \ vskip0.333000 \ baselineskip
      

<210> 5

         \ vskip0.333000 \ baselineskip
      

<211> 220

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ newpage
      

         \ vskip0.333000 \ baselineskip
      

<400> 5

fifteen

         \ vskip0.333000 \ baselineskip
      

<210> 6

         \ vskip0.333000 \ baselineskip
      

<211> 235

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ newpage
      

         \ vskip0.333000 \ baselineskip
      

<400> 6

16

17

         \ vskip0.333000 \ baselineskip
      

<210> 7

         \ vskip0.333000 \ baselineskip
      

<211> 236

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 7

18

19

         \ vskip0.333000 \ baselineskip
      

<210> 8

         \ vskip0.333000 \ baselineskip
      

<211> 219

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 8

twenty

twenty-one

         \ vskip0.333000 \ baselineskip
      

<210> 9

         \ vskip0.333000 \ baselineskip
      

<211> 219

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 9

22

2. 3

         \ vskip0.333000 \ baselineskip
      

<210> 10

         \ vskip0.333000 \ baselineskip
      

<211> 657

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 10

24

         \ vskip0.333000 \ baselineskip
      

<210> 11

         \ vskip0.333000 \ baselineskip
      

<211> 13865

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 11

25

26

27

28

29

         \ vskip0.333000 \ baselineskip
      

<210> 12

         \ vskip0.333000 \ baselineskip
      

<211> 263

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> Homo sapiens

         \ vskip1.000000 \ baselineskip
      

         \ newpage
      

         \ vskip0.333000 \ baselineskip
      

<400> 12

30

         \ vskip0.333000 \ baselineskip
      

<210> 13

         \ vskip0.333000 \ baselineskip
      

<211> 1440

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> mammalian

         \ vskip1.000000 \ baselineskip
      

         \ newpage
      

         \ vskip0.333000 \ baselineskip
      

<400> 13

31

         \ vskip0.333000 \ baselineskip
      

<210> 14

         \ vskip0.333000 \ baselineskip
      

<211> 466

         \ vskip0.333000 \ baselineskip
      

<212> PRT

         \ vskip0.333000 \ baselineskip
      

<213> mammalian

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 14

32

33

3. 4

         \ vskip0.333000 \ baselineskip
      

<210> 15

         \ vskip0.333000 \ baselineskip
      

<211> 27

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 15

\hfill

27gtcatgccat ggcctcaggc actacaa

 \ hfill 

27

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 16

         \ vskip0.333000 \ baselineskip
      

<211> 31

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 16

\hfill

31tgacaagctt attctctgaa ttcccctttc t

 \ hfill 

31

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 17

         \ vskip0.333000 \ baselineskip
      

<211> 47

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 17

\hfill

47gtcatgccat ggccctggtg cctccgtgctt ctgggcactac aaatact

 \ hfill 

47

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 18

         \ vskip0.333000 \ baselineskip
      

<211> 50

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 18

\hfill

50gtcatgccat ggccctggtg cctcgtggtt cttgcggcac tacaaatact

 \ hfill 

fifty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 19

         \ vskip0.333000 \ baselineskip
      

<211> 53

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 19

\hfill

53cgcggatcca ccgccaccag atccaccgcc tccttctctg aattcccctt tct

 \ hfill 

53

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 20

         \ vskip0.333000 \ baselineskip
      

<211> 44

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 20

\hfill

44cgcggatccg gcggtggagg ctcttcaggc actacaaata ctgt

 \ hfill 

44

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 21

         \ vskip0.333000 \ baselineskip
      

<211> 31

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 21

\hfill

31tgacaagctt attctctgaa ttcccctttc t

 \ hfill 

31

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 22

         \ vskip0.333000 \ baselineskip
      

<211> 50

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 22

\hfill

50gtcatgccat ggccctggtg cctcgtggtt cttgcggcac taccaaatact

 \ hfill 

fifty

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 23

         \ vskip0.333000 \ baselineskip
      

<211> 31

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 23

\hfill

31tgacaagctt attcctctgaa ttcccctttc t

 \ hfill 

31

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 24

         \ vskip0.333000 \ baselineskip
      

<211> 44

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 24

\hfill

44gtcatgccat ggccctggtg cctcgtggtt gcacctacaaa tact

 \ hfill 

44

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 25

         \ vskip0.333000 \ baselineskip
      

<211> 34

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 25

\hfill

34tgacaagctt agcattctct gaattcccct ttct

 \ hfill 

3. 4

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 26

         \ vskip0.333000 \ baselineskip
      

<211> 19

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 26

\hfill

19caagttcagc caagaaaac

 \ hfill 

19

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<210> 27

         \ vskip0.333000 \ baselineskip
      

<211> 36

         \ vskip0.333000 \ baselineskip
      

<212> DNA

         \ vskip0.333000 \ baselineskip
      

<213> Artificial sequence

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<220>

         \ vskip0.333000 \ baselineskip
      

<223> Description of artificial sequence: SYNTHETIC PRIMER

         \ vskip1.000000 \ baselineskip
      

         \ vskip0.333000 \ baselineskip
      

<400> 27

\hfill

36acactttatt atcggaaatc ttcagcttca ggaaag

 \ hfill 

36

Claims (47)

1. Composition comprising an amount biologically effective of a Tissue Factor compound of poor coagulation that is at least 100 times less active in coagulation than the original, full-size Fabric Factor, and which has been modified to increase its biological half-life; in the that the deficient coagulation tissue factor compound does not It is attached to an objective part. 2. Composition according to claim 1, for its use to promote coagulation in the tumor vasculature of a animal. 3. Use of a composition, according to the claim 1 or 2, in the manufacture of a medicament for its use in the treatment of vascularized tumors promoting coagulation in the tumor vasculature of an animal. 4. Use of a composition comprising a biologically effective amount of a Tissue Factor compound of poor coagulation in the manufacture of a medicine for its use in the treatment of vascularized tumors promoting coagulation in the tumor vasculature of an animal; in which the Coagulation Factor Factor deficient tissue is like minimum 100 times less active in coagulation than the Tissue Factor original, full size, and not attached to a part objective. 5. Use according to claim 4, wherein the Composite of deficient coagulation tissue factor has been modified to increase its biological half-life. 6. Use according to any of the claims 3, 4 or 5, in which the Clotting Tissue Factor compound deficient has been modified to increase its biological half-life by binding to an inert carrier protein or a molecule non-protein carrier. 7. Use according to claim 6, wherein the Deficient Clotting Fabric Factor compound binds to a albumin or globulin carrier molecule. 8. Use according to claim 6, wherein the Deficient Clotting Fabric Factor compound binds to a inert carrier antibody, or part thereof. 9. Use according to claim 6, wherein the Deficient Clotting Fabric Factor compound binds to a inert carrier IgG antibody, an Fc part of an antibody or is inserted into an inert carrier IgG molecule instead of the domain C_ {H} 3. 10. Use according to any of the claims 3 to 9, wherein the coagulation tissue factor compound deficient is between 100 times and 1,000,000 times less active in coagulation than the original Fabric Factor, in size full. 11. Use according to any of the claims 3 to 10, wherein the Clotting Tissue Factor compound poor is at least 1,000 times less active in coagulation than the original, full-size Fabric Factor. 12. Use according to any of the claims 3 to 11, wherein the coagulation tissue factor compound poor is at least 10,000 times less active in coagulation than the original, full-size Fabric Factor. 13. Use according to any of the claims 3 to 12, wherein the coagulation tissue factor compound poor is at least 100,000 times less active in coagulation than the original, full-size Fabric Factor. 14. Use according to any of the claims 3 to 13, wherein the coagulation tissue factor compound deficient is at least 500,000 times less active in coagulation than the original, full-size Fabric Factor. 15. Use according to any of the claims 3 to 14, wherein the coagulation tissue factor compound deficient is at least 1,000,000 times less active in coagulation than the original Fabric Factor, in size full. 16. Use according to any of the claims 3 to 15, wherein the coagulation tissue factor compound deficient is a mutant tissue factor compound deficient in its ability to activate Factor VII. 17. Use according to claim 16, wherein the Coagulation Factor Factor deficient tissue is a mutant tissue factor compound that includes at least one first mutation in the amino acid region between position 157 and position 167 of SEQ ID No: 1. 18. Use according to claim 17, wherein the Coagulation Factor Factor deficient tissue is a compound of mutant tissue factor in which the Trp in the position 158 is changed to Arg; Being in position 162 is changed to To; Gly at position 164 is changed to Ala; or in which Trp in position 158 is changed to Arg and Ser in position 162 is changed by Ala. 19. Use according to any of the claims 3 to 18, in which the Clotting Tissue Factor compound deficient is a compound of deficient tissue factor for bind to a phospholipid surface. 20. Use according to any of the claims 3 to 19, wherein the coagulation tissue factor compound Poor is a compound of Truncated Tissue Factor. 21. Use according to any of the claims 3 to 20, wherein the coagulation tissue factor compound Poor is a compound of Truncated Tissue Factor of 219 amino acids in size. 22. Use according to any of the claims 3 to 21, wherein the coagulation tissue factor compound Deficient is a compound of Homodimeric Tissue Factor, heterodimeric or polymeric. 23. Use according to any of the claims 3 to 22, wherein the coagulation tissue factor compound Poor is:

(to)

a compound of Mutant Tissue Factor consisting essentially of the sequence of amino acids of SEQ ID No: 8 or SEQ ID No: 9; or

(b)

a compound of Truncated Tissue Factor consisting essentially of the sequence of amino acids of SEQ ID No: 1.

24. Use according to any of the claims 3 to 23, in which the Clotting Tissue Factor compound Deficient is a compound of Human Tissue Factor. 25. Use according to any of the claims 3 to 24, in which the Clotting Tissue Factor compound deficient is a tissue factor compound prepared by recombinant expression 26. Use according to any of the claims 3 to 25, wherein the composition comprises a second compound of Poor coagulation tissue factor. 27. Use according to any of the claims 3 to 26, in combination with a biologically effective amount of at least one Factor VIIa or a Factor VII activator. 28. Use according to claim 27 in combination with Factor VIIa. 29. Use according to claim 28, wherein the Factor VIIa consists essentially of the amino acid sequence from amino acid 61 to amino acid 212 of the sequence of Factor VII polypeptides of SEQ ID No: 14. 30. Use according to any of the claims 27 to 29, wherein the coagulation tissue factor compound deficient is combined with Factor VIIa in a preformed complex of Tissue Factor-Factor VIIa. 31. Use according to any of the claims 3 to 30, in combination with a therapeutically effective amount of an anticancer agent. 32. Use according to claim 31, wherein the Anticancer agent is a chemotherapeutic agent. 33. Use according to claim 32, wherein the Anticancer agent is one among: alkylating agents, antimetabolites, natural products, hormones and antagonists,

         \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {| c |} \ hline
Nitrogen mustards \\\ hline Ethylemoneas and \\ Methylmelamines
\\\ hline Alkyl sulphonate \\\ hline Nitrosoureas \\ Triazines
\\\ hline Folic Acid Analogs \\\ hline Pyrimidine Analogs
\\\ hline
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
      

(continuation)

         \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {| c |} \ hline
Purine Analogs and \\ Related Inhibitors \\\ hline
Vinca alkaloids \\\ hline Epipodophyllotoxins \\\ hline Antibiotics
\\\ hline \\\ hline Enzymes \\\ hline Response Modifiers \\
 Biological \\\ hline Coordination Complexes \\ Platinum \\\ hline
 Antracenediona \\\ hline Urea Substituted \\\ hline Methyl Derivative
hydrazine \\ Adrenocortical \\\ hline Suppressor \\\ hline
Adrenocorticoids \\\ hline Progestin \\\ hline Estrogen
\\\ hline Antiestrogen \\\ hline Androgens \\\ hline Antiandrogen
\\ hormone releasing \\ Gonadotropin \\\ hline
Mechlorethamine (HN2) \\\ hline Cyclophosphamide \\ Ifosfamide
\\\ hline Melphalan (L-sarcolysin) \\\ hline
Chlorambucil \\\ hline Hexamethylmelamine \\\ hline Tiotepa \\\ hline
Busulfan \\\ hline Carmustina (BCNU) \\\ hline Lomustina (CCNU)
\\\ hline Semustina \\ (methyl-CCNU) \\\ hline
Streptozocin \\ (streptozotocin) \\ Decarbazine (DTIC; \\
dimethyltriacenoimide \\ zolecarboxamide) \\\ hline Methotrexate \\
(ametopterin) \\\ hline Fluouracilo \\
(5-fluorouracil; 5-FU) \\
Floxuridine (fluorode- \\ oxyuridine; FUdR)
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
      

(continuation)

         \ catcode` \ # = 12 \ nobreak \ centering \ begin {tabular} {| c |} \ hline
Cytarabine (cytosine \\ arabinoside) \\\ hline Mercaptopurine \\
(6-mercaptopurine; 6-MP) \\\ hline
Thioguanine \\ (6-thioguanine; TG) \\\ hline
Pentostatin \\ (2-deoxicoformicin) \\\ hline
Vinblastine (VLB) \\\ hline Vincristine \\\ hline Etoposide \\
Tertiposide \\\ hline Dactinomycin \\ (actinomycin D) \\\ hline
Daunorubicin \\ (daunomycin; \\ rubidomycin) \\\ hline
Doxorubicin \\ Bleomycin \\\ hline Plicamicin (mitramycin)
\\\ hline Mitomycin (mitomycin C) \\\ hline
L-Asparaginase \\\ hline Interferon alfa \\\ hline
Cisplatín (cis -DDP) \\ Carboplatín \\\ hline Mitoxantrona
\\\ hline Hydroxyurea \\\ hline Procarbacina \\
(N-methylhydrazine, MIH) \\ Mitotane
(o, p '-DDD) \\\ hline Aminoglutethimide
\\\ hline Prednisone (several other preparations are available \\
equivalent) \\\ hline Hydroxyprogesterone \\ caproate \\
Medroxyprogesterone \\ acetate \\ Megestrol acetate \\\ hline
Diethylstilbestrol \\ Etinyl estradiol (\\ other available \\
prepared) \\\ hline Tamoxifen \\\ hline Testosterone propionate \\
 Fluoxymesterone (\\ other preparations are available \\ hline
Flutamide \\ Leuprólido
\\\ hline \ end {tabular} \ par \ vskip.5 \ baselineskip
      

34. Use according to claim 33, wherein the Anticancer agent is etoposide. 35. Use according to claim 31, wherein the anticancer agent is an antibody construct that comprises an antibody or antigen binding region that specifically binds to a component of a tumor cell, tumor vasculature or tumor stroma, the antibody is operatively bound to an agent cytotoxic or a coagulation factor. 36. Use according to claim 35, wherein the anticancer agent is an antibody construct that binds specifically to a component of tumor vasculature or stroma tumor. 37. Use according to claim 35, wherein the anticancer agent is an antibody construct that comprises a cytotoxic agent 38. Use according to claim 35, wherein the anticancer agent is an antibody construct that comprises a coagulation factor 39. Use according to claim 38, wherein the anticancer agent is an antibody construct that comprises Tissue Factor or a derivative of Tissue Factor. 40. Use according to any of the claims 3 to 39, in which the medicine is for use in promoting coagulation in the tumor vasculature associated with a malignant tumor medium-sized or large vascularized of an animal. 41. Use according to any of the claims 3 to 40, in which the medicament is formulated for administration Systemic to the animal. 42. Use according to any of the claims 3 to 41, in which the medicine is formulated for injection intravenous to the animal. 43. Use according to any of the claims 3 to 42, in which the medicament is intended for administration to a human subject 44. Therapeutic case comprising, in a convenient container element:

(to)

A combination biologically effective of a composition according to claim 1 or 2, and at least one among Factor VIIa, an activator of Factor VII or an anticancer agent; or

(b)

A combination biologically effective of an anticancer agent and a compound of Deficient coagulation tissue factor that is at least 100 times less active in coagulation than the original Tissue Factor, full size, and not attached to an objective part.

45. The case according to claim 44, which comprises a biologically effective combination of a composition according to claim 1 or 2, and at least one between Factor VIIa, a Factor VII activator or agent anticancer 46. The case according to claim 44, which comprises a biologically effective combination of an agent anticancer and a coagulation tissue factor compound poor that is at least about 100 times less active in coagulation than the original, full-size Fabric Factor, and does not join an objective part. 47. The case according to claim 44, which comprises a biologically effective combination of (a) a Composite of deficient coagulation tissue factor which is like minimum 100 times less active in coagulation than the Tissue Factor original, full size, and not attached to an objective part; (b) at least one between Factor VIIa or a Factor activator VII; and (c) an anticancer agent.